Loss of production and animal health costs in assessing economic burden of animal disease.

This article focuses on identifying the loss of production and costs (or lack thereof) associated with livestock health as well as animal disease externalities, with the intent to estimate economy-wide burden. It limits its scope to terrestrial livestock and aquaculture, wherein economic burden is predominately determined by market forces. Losses and costs are delineated into both direct losses and costs and indirect losses and costs, as well as ex post costs and ex ante costs. These costs include not only private expenditures but also public expenditures related to the prevention of, treatment of, and response to livestock disease. This distinction is important because a primary role of government is to mitigate externalities. The article then discusses market impacts and investments. Finally, it provides selected examples and illustrative observations and discusses future directions for research and application.

Cet article examine les pertes de production et les coûts associés (ou non) à la santé animale ainsi que les externalités liées aux maladies animales, dans le but d'estimer le fardeau pour l'ensemble de l'économie. L'examen se limite à la production d'animaux terrestres et aquatiques, secteurs où le fardeau économique est principalement déterminé par les forces du marché. Les pertes et les coûts sont répartis en pertes et coûts directs et indirects, ainsi qu'en coûts ex post et ex ante. Ces coûts comprennent non seulement les dépenses privées, mais aussi les dépenses publiques liées à la prévention, au traitement et aux réponses aux maladies des animaux d'élevage. Il s'agit d'une distinction importante car l'une des fonctions premières d'un gouvernement est d'atténuer les externalités. Les auteurs examinent ensuite les impacts sur les marchés et les investissements. Pour conclure, à partir d'exemples choisis et d'observations illustrant leur propos, les auteurs proposent des voies d'exploration pour la recherche et ses applications.

Este artículo se centra en determinar las pérdidas de producción y los costos (o la ausencia de ellos) asociados con las externalidades de la sanidad del ganado y las enfermedades animales, con el objetivo de estimar su impacto en toda la economía. El ámbito del artículo se limita a la ganadería terrestre y la acuicultura, donde el impacto económico está principalmente determinado por las fuerzas del mercado. Las pérdidas y los costos se clasifican en pérdidas y costos directos e indirectos, así como en costos ex post y ex ante. Dichos costos incluyen no solo los gastos privados, sino también los gastos públicos relacionados con la prevención y el tratamiento de las enfermedades del ganado y la respuesta ante estas, una distinción que es importante habida cuenta de que una de las principales funciones del gobierno es mitigar las externalidades. En el artículo se analizan a continuación las repercusiones en el mercado y las inversiones y, por último, se presentan algunos ejemplos y observaciones ilustrativas y se examinan las orientaciones futuras de la investigación y sus aplicaciones.

A Collaborating Centre for animal health economics in the Americas.

Countries in the Americas play a vital role in global livestock and aquaculture production. With international trade of livestock and aquaculture products becoming an important source of income for countries, there has been an increased interest in using animal health economics for advocacy and allocation of resources. This article discusses the development of a new Collaborating Centre for the Economics of Animal Health and the applications of the Global Burden of Animal Diseases (GBADs) framework in the Americas region. Incentives for the increased use of economics in animal health decision-making and examples from the Americas region are examined. The article then discusses the newly formed World Organisation for Animal Health Collaborating Centre in the Americas region. Finally, it reviews two ongoing case studies that are implementing the GBADs framework in Peru and the United States of America.

Les pays des Amériques jouent un rôle crucial dans la production mondiale d'animaux d'élevage, qu'il s'agisse d'espèces terrestres ou aquatiques. Le commerce international des produits issus de l'élevage et de l'aquaculture étant désormais une source importante de revenus pour les pays, la possibilité d'utiliser les concepts de l'économie de la santé animale à des fins de plaidoyer et d'allocation des ressources suscite un intérêt croissant. Les auteurs abordent la désignation d'un nouveau Centre collaborateur pour l'économie de la santé animale ainsi que les applications du programme " Impact mondial des maladies animales " (GBADs) dans la région des Amériques. Ils examinent les avantages d'une meilleure prise en compte de l'économie dans les prises de décision en matière de santé animale, illustrés par quelques exemples concrets relevés dans la région. Ils décrivent ensuite la création du nouveau Centre collaborateur de l'Organisation mondiale de la santé animale dans la région des Amériques. Enfin, ils présentent deux études de cas menées actuellement concernant la mise en oeuvre du programme GBADs respectivement au Pérou et aux Etats-Unis d'Amérique.

Los países de las Américas desempeñan un papel fundamental en la producción ganadera y acuícola mundial. Dado que el comercio internacional de productos ganaderos y acuícolas se ha convertido en una importante fuente de ingresos para los países, el interés por utilizar la economía de la sanidad animal en favor de la promoción y la asignación de recursos ha ido en aumento. En este artículo se analiza la creación de un nuevo Centro colaborador para la economía de la sanidad animal y las aplicaciones del marco del impacto global de las enfermedades animales (GBADs) en la región de las Américas. Se examinan los incentivos para un mayor uso de la economía en la toma de decisiones sobre sanidad animal, así como ejemplos de la región. A continuación, en el artículo se presenta el recién creado Centro colaborador de la Organización Mundial de Sanidad Animal en la región de las Américas. Por último, se examinan dos estudios de casos en los que se está poniendo en práctica el marco del GBADs en Perú y los Estados Unidos de América.

Low Prevalence of Screen-Detected Colorectal Cancer in an Average-Risk Population: The New Normal.

Prior studies have reported the prevalence of colorectal cancer (CRC) in average-risk screening population ages 50-75 to be 0.7%-1.0%.1,2 However, no estimates from studies enrolling individuals undergoing screening colonoscopy have been reported. The experience of ongoing studies enrolling average-risk individuals is that the prevalence rates are substantially lower. A 2020 study from a community-based cohort undergoing CRC screening with fecal immunochemical testing followed by diagnostic colonoscopy reported a CRC prevalence rate of 1.46 per 1000, or 0.15%.3 The aim of our study is to report the screen-detected prevalence of CRC and advanced neoplasia in average-risk asymptomatic individuals from selected academic and community medical centers in the United States, Canada, and Germany and describe associated risk factors.

Global Burden of Animal Diseases: a novel approach to understanding and managing disease in livestock and aquaculture.

Investments in animal health and Veterinary Services can have a measurable impact on the health of people and the environment. These investments require a baseline metric that describes the burden of animal health and welfare in order to justify and prioritise resource allocation and from which to measure the impact of interventions. This paper is part of a process of scientific enquiry in which problems are identified and solutions sought in an inclusive way. It poses the broad question: what should a system to measure the animal disease burden on society look like and what value would it add? Moreover, it aims to do this in such a way as to be accessible by a wide audience, who are encouraged to engage in this debate. Given that farmed animals, including those raised by poor smallholders, are an economic entity, this system should be based on economic principles. These poor farmers are negatively impacted by disparities in animal health technology, which can be addressed through a mixture of supply-led and demand-driven interventions, reinforcing the relevance of targeted financial support from government and non-governmental organisations. The Global Burden of Animal Diseases (GBADs) Programme will glean existing data to measure animal health losses within carefully characterised production systems. Consistent and transparent attribution of animal health losses will enable meaningful comparisons of the animal disease burden to be made between diseases, production systems and countries, and will show how it is apportioned by people's socio-economic status and gender. The GBADs Programme will produce a cloud-based knowledge engine and data portal, through which users will access burden metrics and associated visualisations, support for decisionmaking in the form of future animal health scenarios, and the outputs of wider economic modelling. The vision of GBADs, strengthening the food system for the benefit of society and the environment, is an example of One Health thinking in action.

Les investissements réalisés en santé animale et dans les Services vétérinaires ont un impact mesurable sur la santé des personnes et de l'environnement. Le système de mesure appliqué à ces investissements doit reposer sur un référentiel de base décrivant l'impact de la santé et du bien-être animal de manière à justifier et classer par priorités les ressources allouées et à mesurer les effets des interventions. Les auteurs présentent une étude conduite dans le cadre d'une enquête scientifique destinée à identifier les problèmes et à rechercher des solutions de manière inclusive. L'étude pose la question de savoir à quoi devrait ressembler un système conçu pour mesurer l'impact sur la société des maladies animales, et quelle serait sa valeur ajoutée. En outre, l'étude est conduite de manière à être accessible à une large audience afin d'encourager cette dernière à participer aux discussions. Étant donné que les animaux d'élevage constituent une entité économique, y compris les animaux appartenant à des éleveurs pauvres, le système de mesure doit reposer sur des principes économiques. Les exploitants pratiquant une agriculture de subsistance subissent les effets négatifs des disparités entre les différentes technologies applicables à la santé animale, disparités auxquelles il est possible de remédier par le biais d'interventions associant des mesures dictées par l'offre et par la demande et en renforçant l'efficacité du soutien financier ciblé apporté par les organisations gouvernementales et non gouvernementales. Le Programme « L'impact mondial des maladies animales ¼ (GBADs) aura pour tâche de glaner les données existantes afin de mesurer les pertes associées à la santé animale au sein de systèmes de production qui auront été soigneusement caractérisés au préalable. Grâce à l'élucidation cohérente et transparente des pertes imputables à chaque problème de santé animale, des comparaisons pertinentes pourront être effectuées concernant l'impact des maladies animales par maladies, par systèmes de production et par pays, et la répartition de cet impact dans les populations concernées suivant le statut socio-économique et le genre des intéressés sera mieux comprise. Le Programme GBADs entend créer un moteur de recherche et un portail de données qui seront disponibles sur le Cloud et donneront aux utilisateurs l'accès à des outils de mesure de l'impact des maladies et à d'autres informations présentées sous forme graphique, ainsi qu'à des outils d'aide à la décision sous forme de scénarios prospectifs sur la santé animale et aux résultats d'études plus larges de modélisation économique. La vision du GBADs, renforcer le système de production de denrées alimentaires au profit de la société et de l'environnement, est un exemple de mise en oeuvre du concept Une seule santé.

Las inversiones en sanidad animal y en los Servicios Veterinarios pueden tener un efecto mensurable en la salud de las personas y el medio ambiente. Para efectuar estas inversiones se precisan parámetros que describan y cuantifiquen la situación de partida y el impacto de los problemas de sanidad y bienestar animales, a fin de poder, a partir de ahí, justificar y jerarquizar la asignación de recursos y medir los efectos de las intervenciones. Este artículo, inscrito en un proceso de indagación científica encaminado a detectar problemas y buscar soluciones de forma incluyente, plantea la cuestión general de cómo debería ser y qué valor añadido aportaría un sistema destinado a medir el impacto que imponen a la sociedad las enfermedades animales. Los autores, además, tratan de exponer la cuestión de manera que sea accesible a un público amplio, al que se alienta a participar en este debate. Dado que los animales de granja (incluidos los de pequeñas explotaciones) constituyen una entidad económica, tal sistema debería estar basado en principios económicos. Los productores que trabajan en régimen de subsistencia se ven negativamente afectados por las disparidades existentes en materia de tecnología zoosanitaria, disparidad que cabe corregir con una combinación de intervenciones marcadas por la oferta y otras marcadas por la demanda, dirigiendo así más selectivamente el apoyo económico de entidades gubernamentales y organizaciones no gubernamentales. El programa GBADs (El impacto global de las enfermedades animales) servirá para compilar datos ya existentes con el fin de medir las pérdidas zoosanitarias dentro de sistemas productivos cuidadosamente caracterizados. La atribución coherente y transparente de estas pérdidas zoosanitarias permitirá efectuar comparaciones significativas del impacto que representan las enfermedades animales en el caso de diferentes dolencias, sistemas productivos o países y pondrá de relieve cómo se distribuye este impacto en función del género y la condición socioeconómica de las personas. Por medio del programa GBADs se creará un motor de conocimiento y portal de datos ubicado en la nube que permita al usuario acceder a mediciones del impacto de enfermedades y representaciones gráficas conexas, a herramientas de apoyo a la adopción de decisiones, en forma de hipotéticas situaciones zoosanitarias futuras, y a los resultados de modelizaciones económicas más generales. La aspiración del programa GBADs ­ reforzar el sistema alimentario en beneficio de la sociedad y el medio ambiente ­ constituye un ejemplo de aplicación en la práctica del pensamiento en clave de Una sola salud.

Antagonistic Interactions and Biofilm Forming Capabilities Among Bacterial Strains Isolated from the Egg Surfaces of Lake Sturgeon (Acipenser fulvescens).

Characterization of interactions within a host-associated microbiome can help elucidate the mechanisms of microbial community formation on hosts and can be used to identify potential probiotics that protect hosts from pathogens. Microbes employ various modes of antagonism when interacting with other members of the community. The formation of biofilm by some strains can be a defense against antimicrobial compounds produced by other taxa. We characterized the magnitude of antagonistic interactions and biofilm formation of 25 phylogenetically diverse taxa that are representative of isolates obtained from egg surfaces of the threatened fish species lake sturgeon (Acipenser fulvescens) at two ecologically relevant temperature regimes. Eight isolates exhibited aggression to at least one other isolate. Pseudomonas sp. C22 was found to be the most aggressive strain, while Flavobacterium spp. were found to be one of the least aggressive and the most susceptible genera. Temperature affected the prevalence and intensity of antagonism. The aggressive strains identified also inhibited growth of known fish pathogens. Biofilm formations were observed for nine isolates and were dependent on temperature and growth medium. The most aggressive of the isolates disrupted biofilm formation of two well-characterized isolates but enhanced biofilm formation of a fish pathogen. Our results revealed the complex nature of interactions among members of an egg associated microbial community yet underscored the potential of specific microbial populations as host probiotics.

Animal health economics: an aid to decisionmaking on animal health interventions - case studies in the United States of America.

For animal disease events the outcomes and consequences often remain unclear or uncertain, including the expected changes in benefits (e.g. profit to firms, prices to consumers) and in costs (e.g. response, clean-up). Moreover, the measurement of changes in benefits and costs across alternative interventions used to control animal disease events may be inexact. For instance, the economic consequences of alternative vaccination strategies to mitigate a disease can vary in magnitude due to trade embargoes and other factors. The authors discuss the economic measurement of animal disease outbreaks and interventions and how measurement is used in private and public decision-making. Two illustrative case studies in the United States of America are provided: a hypothetical outbreak of foot and mouth disease in cattle, and the 2014-2015 outbreak of highly pathogenic avian influenza in poultry.

Lors d'un événement sanitaire, les résultats et les conséquences d'une intervention sont souvent incertains ou imprécis, y compris pour ce qui concerne l'évolution attendue des bénéfices (par ex. le profit pour les entreprises ou le prix payé par le consommateur) et des coûts (par ex. le coût de la réponse ou de l'assainissement). De plus, la mesure de l'évolution des bénéfices et des coûts suivant les différentes interventions utilisées pour lutter contre les maladies animales peut s'avérer inexacte. Par exemple, les conséquences économiques de différentes stratégies de vaccination visant à atténuer l'impact d'une maladie peuvent varier en ordre de grandeur du fait des restrictions imposées au commerce suite à la vaccination, ou d'autres facteurs. Les auteurs examinent l'évaluation économique des foyers de maladies animales et des interventions sanitaires ainsi que l'utilisation de ces évaluations dans les prises de décision du secteur privé et public. L'analyse est illustrée par deux études de cas aux États- Unis d'Amérique : l'hypothèse d'un foyer de fièvre aphteuse survenant dans la population bovine, et le foyer d'influenza aviaire hautement pathogène survenu en 2014­2015 chez les volailles.

A menudo los resultados o efectos de ciertos episodios zoosanitarios quedan poco claros o generan incertidumbre, por ejemplo sobre el modo en que en principio modifican los beneficios (réditos para las empresas, precios para el consumidor) y los costos (p.ej. de respuesta o de saneamiento de la explotación). Además, la medición de los cambios que experimenten los costos y beneficios a resultas de distintas intervenciones posibles para combatir un episodio zoosanitario puede resultar inexacta. Por ejemplo: las consecuencias económicas de estrategias alternativas de vacunación para mitigar una enfermedad pueden ser de magnitud variable dependiendo de la existencia de embargos comerciales u otros factores. Los autores examinan la cuantificación económica de los brotes de enfermedades animales y las intervenciones para combatirlos y explican cómo se utilizan esas mediciones para tomar decisiones en los sectores público y privado, ofreciendo como ejemplo casos situados en los Estados Unidos de América: un brote hipotético de fiebre aftosa en el ganado vacuno y el brote de influenza aviar altamente patógena que en 2014 y 2015 afectó a las aves de corral.

Economic Assessment of Zoonotic Diseases: An Illustrative Study of Rift Valley Fever in the United States.

This study evaluates the economic consequences of a Rift Valley Fever outbreak, a virus that spreads from livestock to humans, often through mosquitoes. Developing a 'one health' economic framework, economic impacts on agricultural producers and consumers, government costs of response, costs and disruptions to non-agricultural activities in the epidemiologically impacted region, and human health costs (morbidity and mortality) are estimated. We find the agricultural firms bear most of the negative economic impacts, followed by regional non-agricultural firms, human health and government. Further, consumers of agricultural products benefit from small outbreaks due to bans on agricultural exports.

Bacterial community dynamics during industrial malting, with an emphasis on lactic acid bacteria.

Characterization of the microflora during malting is an essential step towards process management and optimization. Up till now, however, microbial characterization in the malting process has mostly been done using culture-dependent methods, probably leading to biased estimates of microbial diversity. The aim of this study was to characterize the bacterial communities using two culture-independent methods, including Terminal Restriction Fragment Length Polymorphism (T-RFLP) and 454 pyrosequencing, targeting the 16S rRNA gene. Studied samples originated from two harvest years and two malting houses malting the same batch of barley. Besides targeting the entire bacterial community (T-RFLP), emphasis was put on lactic acid bacteria (LAB) (T-RFLP and 454 pyrosequencing). The overall bacterial community richness was limited, but the community structure changed during the process. Zooming in on the LAB community using 454 pyrosequencing revealed a total of 47 species-level operational taxonomic units (OTUs). LAB diversity appeared relatively limited since 88% of the sequences were covered by the same five OTUs (representing members of Weissella, Lactobacillus and Leuconostoc) present in all samples investigated. Fluctuations in the relative abundances of the dominant LAB were observed with the process conditions. In addition, both the year of harvest and malting house influenced the LAB community structure.

Diversity of fish-associated flavobacteria of Michigan.

Flavobacteriosis poses a serious threat to wild and propagated fish stocks alike, accounting for more fish mortality in Michigan and its associated state fish hatcheries than all other pathogens combined. Although this consortium of fish diseases has primarily been attributed to Flavobacterium psychrophilum, F. columnare, and F. branchiophilum, herein we describe a diverse assemblage of Flavobacterium and Chryseobacterium spp. isolates recovered from diseased as well as apparently healthy wild, feral, and farmed fish of Michigan. Among 254 fish-associated flavobacterial isolates recovered from 21 fish species during 2003-2010, 211 were identified as Flavobacterium spp., whereas 43 were identified as Chryseobacterium spp. according to ribosomal RNA partial gene sequencing and phylogenetic analysis. Although F. psychrophilum and F. columnare were indeed associated with multiple fish mortality events, many previously uncharacterized flavobacteria were recovered from systemically infected fish showing overt signs of disease, and in vitro protease assays demonstrated that these isolates were highly proteolytic to multiple substrates that comprise host tissues. Indeed, the majority of the isolates either (1) were most similar to recently described fish-associated Flavobacterium and Chryseobacterium spp. that have never before been reported in North America (e.g., F. oncorhynchi, F. araucananum, C. viscerum, C. piscicola, and C. chaponense) or (2) did not cluster with any described species and most likely represent novel flavobacterial taxa. This study highlights the extreme diversity of flavobacteria that are potentially associated with flavobacteriosis in Michigan.

Assessment of intraradicular bacterial composition by terminal restriction fragment length polymorphism analysis.

BACKGROUND: The aim of the study was to assess the bacterial community structures associated with endodontic infections using terminal restriction fragment length polymorphism (T-RFLP), and to investigate the correlation of whole community profiles with the manifestation of particular clinical features. METHODS: Intraradicular samples were collected from 34 subjects and classified into three study groups based on the observed clinical symptoms: acute (n = 16), sub-acute (n = 8), and asymptomatic (n = 10). Genomic DNA was extracted from each sample, submitted to polymerase chain reaction using a fluorescently labeled 16S ribosomal DNA forward primer, and digested with two tetrameric endonucleases (HhaI and MspI). The terminal restriction fragments (T-RFs) were subsequently discriminated in an automated DNA sequencer, and the results were filtered using a statistics-based criterion. RESULTS: Totals of 138 (HhaI) and 145 (MspI) unique T-RFs were detected (means 13.1 and 11.9) and there was high inter-subject variability in the bacterial assemblages. Odds-ratio analysis unveiled the existence of higher order groups of positively associated T-RFs, restating the concept that intricate ecological relationships may take place in the root canal space. A significantly greater T-RF prevalence was detected in acute cases, suggesting a straight correlation between species richness and spontaneous pain. CONCLUSION: Overall, no T-RFLP profile representing a specific bacterial consortium could be associated with the manifestation of symptoms of endodontic origin.

The Ribosomal Database Project (RDP-II): previewing a new autoaligner that allows regular updates and the new prokaryotic taxonomy.

The Ribosomal Database Project-II (RDP-II) pro-vides data, tools and services related to ribosomal RNA sequences to the research community. Through its website (http://rdp.cme.msu.edu), RDP-II offers aligned and annotated rRNA sequence data, analysis services, and phylogenetic inferences (trees) derived from these data. RDP-II release 8.1 contains 16 277 prokaryotic, 5201 eukaryotic, and 1503 mitochondrial small subunit rRNA sequences in aligned and annotated format. The current public beta release of 9.0 debuts a new regularly updated alignment of over 50 000 annotated (eu)bacterial sequences. New analysis services include a sequence search and selection tool (Hierarchy Browser) and a phylogenetic tree building and visualization tool (Phylip Interface). A new interactive tutorial guides users through the basics of rRNA sequence analysis. Other services include probe checking, phylogenetic placement of user sequences, screening of users' sequences for chimeric rRNA sequences, automated alignment, production of similarity matrices, and services to plan and analyze terminal restriction fragment polymorphism (T-RFLP) experiments. The RDP-II email address for questions or comments is rdpstaff@msu.edu.

Microbial diversity and resistance to copper in metal-contaminated lake sediment.

Contamination of habitats with heavy metals has become a worldwide problem. We describe herein the analysis of lake sediment contaminated with high concentrations of copper as a consequence of mine milling disposal over a 100-year period. Copper concentrations in the sediment were found to vary with depth and ranged from 200 to 5500 ppm. Analysis of the microbial community with T-RFLP identified a minimum of 20 operational taxonomic units (OTU). T-RFLP analysis along a depth profile detected as many as nine shared OTUs across 15 centimeters, suggesting a conservation of community structure over this range. Only two genera, Arthrobacter and Ralstonia, were detected among 50 aerobic copper-resistant isolates cultivated on R2A, one of which (Ralstonia sp.) was characterized by the sequestration of copper, identified by electron diffraction scanning, in growing colonies. Scanning electron microscopy showed changes to the outer envelope of the cells when grown in the presence of copper. The copper-resistant Ralstonia isolates were also resistant to Ni, Cd, and Zn, showing two patterns of phenotypic resistant to these three metals in which either resistance to Zn or Ni was expressed in an isolate but never both.

Application of denaturing gradient gel electrophoresis (DGGE) to study the diversity of marine picoeukaryotic assemblages and comparison of DGGE with other molecular techniques.

We used denaturing gradient gel electrophoresis (DGGE) to study the diversity of picoeukaryotes in natural marine assemblages. Two eukaryote-specific primer sets targeting different regions of the 18S rRNA gene were tested. Both primer sets gave a single band when used with algal cultures and complex fingerprints when used with natural assemblages. The reproducibility of the fingerprints was estimated by quantifying the intensities of the same bands obtained in independent PCR and DGGE analyses, and the standard error of these estimates was less than 2% on average. DGGE fingerprints were then used to compare the picoeukaryotic diversity in samples obtained at different depths and on different dates from a station in the southwest Mediterranean Sea. Both primer sets revealed significant differences along the vertical profile, whereas temporal differences at the same depths were less marked. The phylogenetic composition of picoeukaryotes from one surface sample was investigated by excising and sequencing DGGE bands. The results were compared with an analysis of a clone library and a terminal restriction fragment length polymorphism fingerprint obtained from the same sample. The three PCR-based methods, performed with three different primer sets, revealed very similar assemblage compositions; the same main phylogenetic groups were present at similar relative levels. Thus, the prasinophyte group appeared to be the most abundant group in the surface Mediterranean samples as determined by our molecular analyses. DGGE bands corresponding to prasinophytes were always found in surface samples but were not present in deep samples. Other groups detected were prymnesiophytes, novel stramenopiles (distantly related to hyphochytrids or labyrinthulids), cryptophytes, dinophytes, and pelagophytes. In conclusion, the DGGE method described here provided a reasonably detailed view of marine picoeukaryotic assemblages and allowed tentative phylogenetic identification of the dominant members.

Relationship of hydrogen bioavailability to chromate reduction in aquifer sediments.

Biological Cr(VI) reduction was studied in anaerobic sediments from an aquifer in Norman, Okla. Microcosms containing sediment and mineral medium were amended with various electron donors to determine those most important for biological Cr(VI) reduction. Cr(VI) (about 340 microM) was reduced with endogenous substrates (no donor), or acetate was added. The addition of formate, hydrogen, and glucose stimulated Cr(VI) reduction compared with reduction in unamended controls. From these sediments, an anaerobic Cr(VI)-utilizing enrichment was obtained that was dependent upon hydrogen for both growth and Cr(VI) reduction. No methane was produced by the enrichment, which reduced about 750 microM Cr(VI) in less than six days. The dissolved hydrogen concentration was used as an indicator of the terminal electron accepting process occurring in the sediments. Microcosms with sediments, groundwater, and chromate metabolized hydrogen to a concentration below the detection limits of the mercury vapor gas chromatograph. In microcosms without chromate, the hydrogen concentration was about 8 nM, a concentration comparable to that under methanogenic conditions. When these microcosms were amended with 500 microM Cr(VI), the dissolved hydrogen concentration quickly fell below the detection limits. These results showed that the hydrogen concentration under chromate-reducing conditions became very low, as low as that reported under nitrate- and manganese-reducing conditions, a result consistent with the free energy changes for these reactions. The utilization of formate, lactate, hydrogen, and glucose as electron donors for Cr(VI) reduction indicates that increasing the availability of hydrogen results in a greater capacity for Cr(VI) reduction. This conclusion is supported by the existence of an enrichment dependent upon hydrogen for growth and Cr(VI) reduction.

Terminal restriction fragment length polymorphism analysis program, a web-based research tool for microbial community analysis.

Rapid analysis of microbial communities has proven to be a difficult task. This is due, in part, to both the tremendous diversity of the microbial world and the high complexity of many microbial communities. Several techniques for community analysis have emerged over the past decade, and most take advantage of the molecular phylogeny derived from 16S rRNA comparative sequence analysis. We describe a web-based research tool located at the Ribosomal Database Project web site (http://www.cme.msu.edu/RDP/html/analyses. html) that facilitates microbial community analysis using terminal restriction fragment length polymorphism of 16S ribosomal DNA. The analysis function (designated TAP T-RFLP) permits the user to perform in silico restriction digestions of the entire 16S sequence database and derive terminal restriction fragment sizes, measured in base pairs, from the 5' terminus of the user-specified primer to the 3' terminus of the restriction endonuclease target site. The output can be sorted and viewed either phylogenetically or by size. It is anticipated that the site will guide experimental design as well as provide insight into interpreting results of community analysis with terminal restriction fragment length polymorphisms.

Visualization and enumeration of marine planktonic archaea and bacteria by using polyribonucleotide probes and fluorescent in situ hybridization.

Fluorescent in situ hybridization (FISH) using rRNA-specific oligonucleotide probes has emerged as a popular technique for identifying individual microbial cells. In natural samples, however, the signal derived from fluor-labeled oligonucleotide probes often is undetectable above background fluorescence in many cells. To circumvent this difficulty, we applied fluorochrome-labeled polyribonucleotide probes to identify and enumerate marine planktonic archaea and bacteria. The approach greatly enhanced the sensitivity and applicability of FISH with seawater samples, allowing confident identification and enumeration of planktonic cells to ocean depths of 3,400 m. Quantitative whole-cell hybridization experiments using these probes accounted for 90 to 100% of the total 4',6-diamidino-2-phenylindole (DAPI)-stained cells in most samples. As predicted in a previous study (R. Massana, A. E. Murray, C. M. Preston, and E. F. DeLong, Appl. Environ. Microbiol. 63:50-56, 1997), group I and II marine archaea predominate in different zones in the water column, with maximal cell densities of 10(5)/ml. The high cell densities of archaea, extending from surface waters to abyssal depths, suggest that they represent a large and significant fraction of the total picoplankton biomass in coastal ocean waters. The data also show that the vast majority of planktonic prokaryotes contain significant numbers of ribosomes, rendering them easily detectable with polyribonucleotide probes. These results imply that the majority of planktonic cells visualized by DAPI do not represent lysed cells or "ghosts," as was suggested in a previous report.

Terminal restriction fragment length polymorphism (T-RFLP): an emerging method for characterizing diversity among homologous populations of amplification products.

Terminal restriction fragment length polymorphism is a recent molecular approach that can assess subtle genetic differences between strains as well as provide insight into the structure and function of microbial communities. The technique has both high sensitivity and throughput making it ideal for comparative analyses.

Parallel and divergent genotypic evolution in experimental populations of Ralstonia sp.

Genetic rearrangements within a population of bacteria were analyzed to understand the degree of divergence occurring after experimental evolution. We used 18 replicate populations founded from Ralstonia sp. strain TFD41 that had been propagated for 1,000 generations with 2,4-dichlorophenoxyacetic acid (2,4-D) as the carbon source. Genetic divergence was examined by restriction fragment length polymorphism analysis of the incumbent plasmid that carries the 2,4-D catabolic genes and by amplification of random regions of the genome via PCR. In 18 evolved clones examined, we observed duplication within the plasmid, including the tfdA gene, which encodes a 2,4-D dioxygenase that catalyzes the first step in the 2,4-D catabolic pathway. In 71 of 72 evolved clones, a common 2.4-kb PCR product was lost when genomic fingerprints produced by PCR amplification using degenerate primers based on repetitive extragenic palindromic (REP) sequences (REP-PCR) were compared. The nucleotide sequence of the 2.4-kb PCR product has homology to the TRAP (tripartite ATP-independent periplasmic) solute transporter gene family. Hybridization of the 2. 4-kb REP-PCR product from the ancestor to genomic DNA from the evolved populations showed that the loss of the PCR product resulted from deletions in the genome. Deletions in the plasmid and presence and/or absence of other REP-PCR products were also found in these clones but at much lower frequencies. The common and uncommon genetic changes observed show that both parallel and divergent genotypic evolution occurred in replicate populations of this bacterium.

Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA.

A quantitative molecular technique was developed for rapid analysis of microbial community diversity in various environments. The technique employed PCR in which one of the two primers used was fluorescently labeled at the 5' end and was used to amplify a selected region of bacterial genes encoding 16S rRNA from total community DNA. The PCR product was digested with restriction enzymes, and the fluorescently labeled terminal restriction fragment was precisely measured by using an automated DNA sequencer. Computer-simulated analysis of terminal restriction fragment length polymorphisms (T-RFLP) for 1,002 eubacterial sequences showed that with proper selection of PCR primers and restriction enzymes, 686 sequences could be PCR amplified and classified into 233 unique terminal restriction fragment lengths or "ribotypes." Using T-RFLP, we were able to distinguish all bacterial strains in a model bacterial community, and the pattern was consistent with the predicted outcome. Analysis of complex bacterial communities with T-RFLP revealed high species diversity in activated sludge, bioreactor sludge, aquifer sand, and termite guts; as many as 72 unique ribotypes were found in these communities, with 36 ribotypes observed in the termite guts. The community T-RFLP patterns were numerically analyzed and hierarchically clustered. The pattern derived from termite guts was found to be distinctly different from the patterns derived from the other three communities. Overall, our results demonstrated that T-RFLP is a powerful tool for assessing the diversity of complex bacterial communities and for rapidly comparing the community structure and diversity of different ecosystems.