A case for an active eukaryotic marine biosphere during the Proterozoic era.

The microfossil record demonstrates the presence of eukaryotic organisms in the marine ecosystem by about 1,700 million years ago (Ma). Despite this, steranes, a biomarker indicator of eukaryotic organisms, do not appear in the rock record until about 780 Ma in what is known as the "rise of algae." Before this, it is argued that eukaryotes were minor ecosystem members, with prokaryotes dominating both primary production and ecosystem dynamics. In this view, the rise of algae was possibly sparked by increased nutrient availability supplying the higher nutrient requirements of eukaryotic algae. Here, we challenge this view. We use a size-based ecosystem model to show that the size distribution of preserved eukaryotic microfossils from 1,700 Ma and onward required an active eukaryote ecosystem complete with phototrophy, osmotrophy, phagotrophy, and mixotrophy. Model results suggest that eukaryotes accounted for one-half or more of the living biomass, with eukaryotic algae contributing to about one-half of total marine primary production. These ecosystems lived with deep-water phosphate levels of at least 10% of modern levels. The general lack of steranes in the pre-780-Ma rock record could be a result of poor preservation.

Validation of a real-time PCR based method for detection of Clostridium botulinum types C, D and their mosaic variants C-D and D-C in a multicenter collaborative trial.

Two real-time PCR arrays based on the GeneDisc(®) cycler platform (Pall-GeneDisc Technologies) were evaluated in a multicenter collaborative trial for their capacity to specifically detect and discriminate Clostridium botulinum types C, D and their mosaic variants C-D and D-C that are associated with avian and mammalian botulism. The GeneDisc(®) arrays developed as part of the DG Home funded European project 'AnibioThreat' were highly sensitive and specific when tested on pure isolates and naturally contaminated samples (mostly clinical specimen from avian origin). Results of the multicenter collaborative trial involving eight laboratories in five European Countries (two laboratories in France, Italy and The Netherlands, one laboratory in Denmark and Sweden), using DNA extracts issued from 33 pure isolates and 48 naturally contaminated samples associated with animal botulism cases, demonstrated the robustness of these tests. Results showed a concordance among the eight laboratories of 99.4%-100% for both arrays. The reproducibility of the tests was high with a relative standard deviation ranging from 1.1% to 7.1%. Considering the high level of agreement achieved between the laboratories these PCR arrays constitute robust and suitable tools for rapid detection of C. botulinum types C, D and mosaic types C-D and D-C. These are the first tests for C. botulinum C and D that have been evaluated in a European multicenter collaborative trial.

Multi-site-specific 16S rRNA methyltransferase RsmF from Thermus thermophilus.

Cells devote a significant effort toward the production of multiple modified nucleotides in rRNAs, which fine tune the ribosome function. Here, we report that two methyltransferases, RsmB and RsmF, are responsible for all four 5-methylcytidine (m(5)C) modifications in 16S rRNA of Thermus thermophilus. Like Escherichia coli RsmB, T. thermophilus RsmB produces m(5)C967. In contrast to E. coli RsmF, which introduces a single m(5)C1407 modification, T. thermophilus RsmF modifies three positions, generating m(5)C1400 and m(5)C1404 in addition to m(5)C1407. These three residues are clustered near the decoding site of the ribosome, but are situated in distinct structural contexts, suggesting a requirement for flexibility in the RsmF active site that is absent from the E. coli enzyme. Two of these residues, C1400 and C1404, are sufficiently buried in the mature ribosome structure so as to require extensive unfolding of the rRNA to be accessible to RsmF. In vitro, T. thermophilus RsmF methylates C1400, C1404, and C1407 in a 30S subunit substrate, but only C1400 and C1404 when naked 16S rRNA is the substrate. The multispecificity of T. thermophilus RsmF is potentially explained by three crystal structures of the enzyme in a complex with cofactor S-adenosyl-methionine at up to 1.3 A resolution. In addition to confirming the overall structural similarity to E. coli RsmF, these structures also reveal that key segments in the active site are likely to be dynamic in solution, thereby expanding substrate recognition by T. thermophilus RsmF.

Gross and histopathological evaluation of umbilical outpouchings in pigs.

Clinical presentations of umbilical outpouchings (UOs) in pigs cover a variety of pathological manifestations. Pigs with UOs often do not reach the abattoir as they die due to complications or are euthanized for welfare concerns. The primary objective was to characterize the gross appearance of UOs in pigs with respect to the different types of pathological manifestations. Also the association between the pathological manifestation and presence of a wound on the UO was evaluated. Pigs (in different age groups, n = 444) with an UO were sampled in Denmark from different locations (two herds and at an abattoir) and examined post mortem. Tissue samples from animals with an enterocystoma or internal umbilical proliferations were collected for histological and immunohistochemical characterization. Hernia umbilicalis was the most frequent cause (72%, n = 320) of the UOs. It was the only diagnosis in 57% (n = 254) of the pigs, and in 15% (n = 66) of the pigs the hernia appeared in combination with other manifestations. Thus, 28% (n = 124) of the pigs were diagnosed with an enterocystoma, internal umbilical proliferations, subcutaneous abscess/ fibrosis or another diagnosis, presented alone or in combination. The distribution of diagnoses varied in the different age groups. Overall, 38% (110/291) of the pigs presented a wound on the UOs post mortem. The age of the pigs confounded the relation between the pathological manifestation and the presence of a wound. The odds that an UO had a wound were lower among pigs with a subcutaneous abscess/ fibrosis compared to pigs diagnosed with an umbilical hernia or enterocystoma (OR, 0.3; 95% Cl, 0.1-0.7). The odds of wounds were higher among weaners (OR, 4.3; 95% Cl 2.3-8.3) and finishers (OR, 6.5; 95% Cl, 3.4-12.7) compared with piglets from the farrowing unit. The area of wounds ranged from 0.03 to 78.5 cm2 and increased with age (P < 0.001). Histologically and immunohistochemically the enterocystomas and internal umbilical proliferations seemed to be lined with mesothelial cells and both had a content comparable with mesenchymal embryonic connective tissue. However, only the cavities of the enterocystomas were also lined with mesothelial cells. In conclusion, UOs in pigs are caused by complex pathological conditions with hernia umbilicalis as the dominating diagnosis. Knowledge clarifying the different pathological manifestations causing an UO and the presence of wounds on the UOs is essential for future prevention strategies.

Survival of pigs with different characteristics of umbilical outpouching in a prospective cohort study of Danish pigs.

Umbilical hernia and other conditions clinically evident as umbilical outpouchings (UOs) affect the welfare and economy in Danish pig production. The objectives of the current study were to characterize the associations between 1) time of detection of the UOs and the odds of dying before scheduled slaughter; 2) time of death, irrespective of the cause, and clinical signs of the UOs, i.e. general condition, size, reducibility, form and skin-color of the UOs; and 3) occurrence of wounds on the UOs and clinical signs: general condition, size, reducibility, form and skin-color. A cohort of Danish conventional pigs with UOs (n = 255) were followed from the detection of an UO until spontaneous death, euthanization or slaughter of the pig. The pigs were clinically examined once a month, and when pigs with an UO died spontaneously, were euthanized or slaughtered, the causes and date of death were recorded. The effects of the clinical manifestations on overall survival were assessed using a Cox proportional hazards model. In total 57 % of the pigs died spontaneously or were euthanized before slaughter. The median age of spontaneous death or euthanasia was 85 days. The UOs were detected at different ages, with half of the pigs (52 %) detected in the farrowing section. No significant association was found between death before scheduled slaughter and the time of detection. Three different clinical manifestations were found to have a prognostic value for overall survival until slaughter, i.e. skin-color of the UO, a general condition of the pig and the size of the UO. An interaction was present between the size and the skin-color of the UO. Wounds on the UO were the most frequent complication resulting in euthanasia (37 %). The odds for developing a wound on the UO were higher for pigs in a general bad condition compared to pigs in a good condition (OR, 5.4; 95 % CL 2.5-11.3), and for pigs with an UO large in size compared to pigs with a small UO (OR, 4.8; 95 % CL 3.0-7.5). The identification of prognostic clinical signs in pigs with an UO is useful in the assessment and decision-making in relation to the future prospects of pigs with UOs.

Indications for and clinical impact of repeat endoscopic ultrasound.

OBJECTIVE: The value of repeating endoscopic ultrasound (EUS) is seldom described. This study evaluates a patient population in which EUS was repeated. MATERIAL AND METHODS: This was a retrospective study of patients who between January 2002 and December 2006 had an EUS scan performed; this EUS scan (re-EUS) was the second or more EUS scan performed. RESULTS: Over the study period, the department performed 3024 EUS procedures, of which 561 investigations were defined as re-EUS. According to defined exclusion criteria, 244 procedures were not analyzed further. The study group thus consisted of 317 procedures (242 patients). In 163 cases (126 patients), re-EUS was planned by the endosonographer for control of an undetermined lesion. The first re-EUS scan performed changed the further management in 91 of 126 patients (72%). Sensitivity and specificity of re-EUS regarding pancreatic cancer were 0.65 and 1.00, respectively. Re-EUS was performed in 82 cases (77 patients) where no re-investigation had been planned at the initial EUS scan but worsening of symptoms or new findings of other imaging procedures had led to an additional EUS scan. Thirteen of these patients (17%) proved to have pancreatic cancer. In 62 cases (57 patients) re-EUS and EUS-guided fine-needle aspiration (FNA) had been planed in order to confirm the suspicion of malignant disease. Following re-EUS and EUS-FNA, 40 of these patients could be referred for either oncology or surgery. In the remaining 10 cases, re-EUS was performed for miscellaneous indications. CONCLUSION: Re-EUS has a substantial clinical impact on the further management of the patient.

Turoctocog alfa is safe for the treatment of Indian patients with hemophilia A: Guardian 10 trial results.

BACKGROUND: Hemophilia A is an X chromosome-linked bleeding disorder caused by the deficiency of coagulation factor VIII (FVIII). The majority of the Indian population with hemophilia A use plasma-derived clotting factors and, in some instances, fresh frozen plasma and cryoprecipitate. Safer and more efficient treatment options are needed for this group of patients. OBJECTIVES: To assess the safety of turoctocog alfa, a third-generation recombinant FVIII molecule, for the treatment and prophylaxis of bleeding episodes in previously treated Indian patients with moderate or severe hemophilia A. PATIENTS/METHODS: This single-country, multicenter, open-label, nonrandomized trial enrolled 60 patients who received prophylactic treatment with turoctocog alfa for 8 weeks, which corresponded to a minimum of 20 exposure days. Confirmed development of FVIII inhibitors during the 8-week treatment period was evaluated. Other assessments included frequencies of adverse drug reactions (ARs), serious adverse reactions, drug-related allergic reactions, and infusion reactions during the 12-week period after the first treatment; hemostatic effect of turoctocog alfa for the treatment of bleeding episodes; and total annualized dose of turoctocog alfa administered during the 8-week treatment period. RESULTS: No incidence of FVIII inhibitors was detected. No safety concerns such as ARs, serious ARs, or drug-related allergic reactions were noted. The hemostatic success rate for the treatment of bleeding episodes with turoctocog alfa was 81.6%. CONCLUSIONS: The trial results demonstrated that turoctocog alfa is a safe treatment option for the prophylaxis and treatment of bleeding episodes in previously treated adolescent and adult patients with hemophilia A in the Indian population.

Functional oral intake and time to reach unrestricted dieting for patients with traumatic brain injury.

OBJECTIVES: To investigate the status of functional oral intake for patients with severe traumatic brain injury (TBI) and time to return to unrestricted dieting; and to investigate whether severity of brain injury is a predictor for unrestricted dieting. DESIGN: Observational retrospective cohort study. SETTING: Subacute rehabilitation department, university hospital. PARTICIPANTS: Patients age 16 to 65 years (N=173) with severe TBI (posttraumatic amnesia from 7d to >6 mo) admitted over a 5-year period. Patients are transferred to the brain injury unit as soon as they ventilate spontaneously. INTERVENTION: Facial oral tract therapy. MAIN OUTCOME MEASURE: Unrestricted dieting assessed by the Functional Oral Intake Scale (FOIS). RESULTS: We found that 93% of all patients had problems with functional oral intake at admission. Within 126 days of rehabilitation, 64% recovered to unrestricted dieting before discharge. The chance of returning to total oral diet depends on the severity of the brain injury and can be predicted by Glasgow Coma Scale (GCS; measured the day after cessation of sedation; Wald chi(2)=42.78, P<.01), Rancho Los Amigos Scale (RLAS) level (Wald chi(2)=11.84, P=.01), FIM instrument (Wald chi(2)=44.40, P<.01), and FOIS score at admission (Wald chi(2)=82.93, P<.01). CONCLUSIONS: Impairment in functional oral intake was found to be very common for patients with severe TBI admitted to a subacute rehabilitation department. For those who recovered during hospital rehabilitation, return to unrestricted dieting happened within 126 days of rehabilitation. The chance of returning to unrestricted dieting depends on the severity of the brain injury and can be predicted by GCS score, RLAS level, FIM score, and functional oral intake at admission. These results are important when planning rehabilitation, giving information to patients and relatives, and designing efficacy studies of facial oral tract therapy, which are highly recommended.

The association of functional oral intake and pneumonia in patients with severe traumatic brain injury.

OBJECTIVES: To investigate the incidence and onset time of pneumonia for patients with severe traumatic brain injury (TBI) in the early phase of rehabilitation and to identify parameters associated with the risk of pneumonia. DESIGN: Observational retrospective cohort study. SETTING: Subacute rehabilitation department in a university hospital in Denmark. PARTICIPANTS: Patients (N=173) aged 16 to 65 years with severe TBI who were admitted during a 5-year period. Patients are transferred to the brain injury unit as soon as they ventilate spontaneously. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURE: Pneumonia. RESULTS: Twenty-seven percent of the patients admitted to the brain injury unit were in treatment for pneumonia; pneumonia developed in 12% of the patients during rehabilitation; the condition occurred within 19 days of admission in all but 1 patient. Of these patients, 81% received nothing by mouth. Three factors identified patients at highest risk of pneumonia: Glasgow Coma Scale score less than 9 (1 day after cessation of sedation); Rancho Los Amigos Scale score less than 3 (on admission); and no oral intake on admission. Having a tracheotomy tube and/or feeding tube was also associated with a higher occurrence of pneumonia. CONCLUSIONS: Among patients with severe TBI, 27% had pneumonia at transfer from the intensive care unit. Pneumonia developed in only 12% of the participants during rehabilitation. Patients with a low level of consciousness and patients with a tracheotomy tube or feeding tube had a higher likelihood of pneumonia.

Effects of pre-treatment technologies on quantity and quality of source-sorted municipal organic waste for biogas recovery.

Source-sorted municipal organic waste collected from different dwelling types in five Danish cities and pre-treated at three different plants was sampled and characterized several times during one year to investigate the origin of any differences in composition of the pre-treated waste introduced by city, pre-treatment technology, dwelling type or annual season. The investigated pre-treatment technologies were screw press, disc screen and shredder+magnet. The average quantity of pre-treated organic waste (biomass) produced from the incoming waste varied between the investigated pre-treatment technologies: 59%, 66% and 98% wet weight, respectively (41%, 34% and 2% reject, respectively). The pre-treatment technologies showed differences with respect to distribution of the chemical components in the waste between the biomass and the rejected material (reject), especially for dry matter, ash, collection bag material (plastic or paper) and easily degradable organic matter. Furthermore, the particle size of the biomass was related to the pre-treatment technology. The content of plastic in the biomass depended both on the actual collection bag material used in the system and the pre-treatment technology. The sampled reject consisted mostly of organic matter. For cities using plastic bags for the source-separated organic waste, the expected content of plastic in the reject was up to 10% wet weight (in some cases up to 20%). Batch tests for methane potential of the biomass samples showed only minor variations caused by the factors city, pre-treatment technology, dwelling type and season when based on the VS content of the waste (overall average 459STPm(3)/tVS). The amount of methane generated from 1t of collected waste was therefore mainly determined by the efficiency of the chosen pre-treatment technology described by the mass distribution of the incoming waste between biomass and reject.

Methane yield in source-sorted organic fraction of municipal solid waste.

Treating the source-separated organic fraction of municipal solid waste (SS-OFMSW) by anaerobic digestion is considered by many municipalities in Europe as an environmentally friendly means of treating organic waste and simultaneously producing methane gas. Methane yield can be used as a parameter for evaluation of the many different systems that exist for sorting and pre-treating waste. Methane yield from the thermophilic pilot scale digestion of 17 types of domestically SS-OFMSW originating from seven full-scale sorting systems was found. The samples were collected during 1 year using worked-out procedures tested statistically to ensure representative samples. Each waste type was identified by its origin and by pre-sorting, collection and pre-treatment methods. In addition to the pilot scale digestion, all samples were examined by chemical analyses and methane potential measurements. A VS-degradation rate of around 80% and a methane yield of 300-400Nm(3) CH(4)/ton VS(in) were achieved with a retention time of 15 days, corresponding to approximately 70% of the methane potential. The different waste samples gave minor variation in chemical composition and thus also in methane yield and methane potential. This indicates that sorting and collection systems in the present study do not significantly affect the amount of methane produced per VS treated.

Composition of source-sorted municipal organic waste collected in Danish cities.

Source-sorted municipal organic waste from different dwelling types in five Danish cities was sampled during one year. The samples were from permanent, full-scale systems or temporary, experimental systems for collection of source-sorted municipal organic waste. Pre-treatment of the organic waste prior to biological treatment was used in all cities to remove foreign objects and provide size reduction. All sampling was performed after pre-treatment in order to obtain more homogeneous and representative samples. The sampling included both the pre-treated waste and the reject from the pre-treatment allowing for estimation of the composition of the original waste. A total of 40 waste samples were chemically characterised with respect to 15 parameters. The waste generally consisted of around 88% VS of which an average of 80% was easily degradable. The average content of N, P and K in the dry matter of the organic waste was 2.5%, 0.4% and 0.9%, respectively. A general analysis of variance was applied to show the influence of the collection system, dwelling type and annual season on the waste composition. The content of plastic and crude fibres in the waste differed the most among the samples, probably due to use of different bag types (plastic and paper) in the different collection systems. Variations in the ash content and the calorific value might be explained by differences in the sorting instructions (whether soil and cat litter are allowed in the organic fraction). Significant seasonal variations were seen for ash, S and Cl. Dwelling type showed no statistically significant influence on any waste components. A test for uniform distribution of the p-values from the analysis of variance (Kolmogorov-Smirnov test) showed that the overall composition of the collected waste was strongly affected by the collection system (city) and season, while dwelling type had no significant influence.

Methane production during storage of anaerobically digested municipal organic waste.

Anaerobic digestion of source-separated municipal organic waste is considered feasible in Denmark. The limited hydraulic retention in the biogas reactor (typically 15 d) does not allow full degradation of the organic waste. Storage of anaerobically digested municipal organic waste can therefore be a source of methane (CH4) emission that may contribute significantly to the potential global warming impact from the waste treatment system. This study provides a model for quantifying the CH4 production from stored co-digested municipal organic waste and estimates the production under typical Danish climatic conditions, thus quantifying the potential global warming impact from storage of the digested municipal organic waste before its use on agricultural land. Laboratory batch tests on CH4 production as well as temperature measurements in eight full-scale storage tanks provided data for developing a model estimating the CH4 production in storage tanks containing digested municipal organic waste. The temperatures measured in separate storage tanks on farms receiving digested slurry were linearly correlated with air temperature. In storage tanks receiving slurry directly from biogas reactors, significantly higher temperatures were measured due to the high temperatures of the effluent from the reactor. Storage tanks on Danish farms are typically emptied in April and have a constant inflow of digested material. During the warmest months the content of digested material is therefore low, which limits the yearly CH4 production from storage.

Assessment of sampling and chemical analysis of source-separated organic household waste.

The quality of the waste sampling procedure and chemical analysis was evaluated in a research program on characterization of organic waste obtained after disc screening of source-separated organic household waste. The sampling procedures focused on a truckload of waste and involved several steps of subsampling including shredding, mixing, blending, high-speed-blending, drying and milling prior to analysis of the organic waste with respect to ash content, crude fibers, crude fat, crude protein, sugar, starch, enzyme-digestible organic matter, P, N, C, H, S and calorific value. The statistical evaluation of the procedures involved 10 samples of the same truckload of waste obtained by splitting the sample at each level in the procedure according to a staggered, incomplete nested statistical design. Furthermore, one sample was analysed six times over a period of approximately one year. The statistical evaluation showed that no single step in the sampling procedure contributed with excessive variance and that the variance caused by the sampling procedure was approximately the same as the variance in the chemical analysis observed over a year. The variance varied with the analytical parameter but for most parameters the uncertainty was satisfactorily low (of the order of 3-10% expressed as the relative standard deviation, which is considered to be satisfactory for waste characterization).

Method for determination of methane potentials of solid organic waste.

A laboratory procedure is described for measuring methane potentials of organic solid waste. Triplicate reactors with 10 grams of volatile solids were incubated at 55 degrees C with 400 ml of inoculum from a thermophilic biogas plant and the methane production was followed over a 50-day period by regular measurements of methane on a gas chromatograph. The procedure involves blanks as well as cellulose controls. Methane potentials have been measured for source-separated organic household waste and for individual waste materials. The procedure has been evaluated regarding practicality, workload, detection limit, repeatability and reproducibility as well as quality control procedures. For the source-separated organic household waste a methane potential of 495 ml CH4/g VS was found. For fat and oil a lag-phase of several days was seen. The protein sample was clearly inhibited and the maximal methane potential was therefore not achieved. For paper bags, starch and glucose 63, 84 and 94% of the theoretical methane potential was achieved respectively. A detection limit of 72.5 ml CH4/g VS was calculated from the results. This is acceptable, since the methane potential of the tested waste materials was in the range of 200-500 ml CH4/g VS. The determination of methane potentials is a biological method subject to relatively large variation due to the use of non-standardized inoculum and waste heterogeneity. Therefore, procedures for addressing repeatability and reproducibility are suggested.

Evaluation of direct 16S rDNA sequencing as a metagenomics-based approach to screening bacteria in bottled water.

Deliberate or accidental contamination of food, feed, and water supplies poses a threat to human health worldwide. A rapid and sensitive detection technique that could replace the current labor-intensive and time-consuming culture-based methods is highly desirable. In addition to species-specific assays, such as PCR, there is a need for generic methods to screen for unknown pathogenic microorganisms in samples. This work presents a metagenomics-based direct-sequencing approach for detecting unknown microorganisms, using Bacillus cereus (as a model organism for B. anthracis) in bottled water as an example. Total DNA extraction and 16S rDNA gene sequencing were used in combination with principle component analysis and multicurve resolution to study detection level and possibility for identification. Results showed a detection level of 10(5) to 10(6) CFU/L. Using this method, it was possible to separate 2 B. cereus strains by the principal component plot, despite the close sequence resemblance. A linear correlation between the artificial contamination level and the relative amount of the Bacillus artificial contaminant in the metagenome was observed, and a relative amount value above 0.5 confirmed the presence of Bacillus. The analysis also revealed that background flora in the bottled water varied between the different water types that were included in the study. This method has the potential to be adapted to other biological matrices and bacterial pathogens for fast screening of unknown bacterial threats in outbreak situations.

Metagenomic detection methods in biopreparedness outbreak scenarios.

In the field of diagnostic microbiology, rapid molecular methods are critically important for detecting pathogens. With rapid and accurate detection, preventive measures can be put in place early, thereby preventing loss of life and further spread of a disease. From a preparedness perspective, early detection and response are important in order to minimize the consequences. During the past 2 decades, advances in next-generation sequencing (NGS) technology have changed the playing field of molecular methods. Today, it is within reach to completely sequence the total microbiological content of a clinical sample, creating a metagenome, in a single week of laboratory work. As new technologies emerge, their dissemination and capacity building must be facilitated, and criteria for use, as well as guidelines on how to report results, must be established. This article focuses on the use of metagenomics, from sample collection to data analysis and to some extent NGS, for the detection of pathogens, the integration of the technique in outbreak response systems, and the risk-based evaluation of sample processing in routine diagnostics labs. The article covers recent advances in the field, current debate, gaps in research, and future directions. Examples of metagenomic detection, as well as possible applications of the methods, are described in various biopreparedness outbreak scenarios.

In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences.

Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen that is relatively common throughout the world and may cause life threatening diseases in animals and humans. There are many PCR-based assays in use for the detection of B. anthracis. While most of the developed assays rely on unique markers present on virulence plasmids pXO1 and pXO2, relatively few assays incorporate chromosomal DNA markers due to the close relatedness of B. anthracis to the B. cereus group strains. For the detection of chromosomal DNA, different genes have been used, such as BA813, rpoB, gyrA, plcR, S-layer, and prophage-lambda. Following a review of the literature, an in silico analysis of all signature sequences reported for identification of B. anthracis was conducted. Published primer and probe sequences were compared for specificity against 134 available Bacillus spp. genomes. Although many of the chromosomal targets evaluated are claimed to be specific to B. anthracis, cross-reactions with closely related B. cereus and B. thuringiensis strains were often observed. Of the 35 investigated PCR assays, only 4 were 100% specific for the B. anthracis chromosome. An interlaboratory ring trial among five European laboratories was then performed to evaluate six assays, including the WHO recommended procedures, using a collection of 90 Bacillus strains. Three assays performed adequately, yielding no false positive or negative results. All three assays target chromosomal markers located within the lambdaBa03 prophage region (PL3, BA5345, and BA5357). Detection limit was further assessed for one of these highly specific assays.

Animal botulism outcomes in the AniBioThreat project.

Botulism disease in both humans and animals is a worldwide concern. Botulinum neurotoxins produced by Clostridium botulinum and other Clostridium species are the most potent biological substances known and are responsible for flaccid paralysis leading to a high mortality rate. Clostridium botulinum and botulinum neurotoxins are considered potential weapons for bioterrorism and have been included in the Australia Group List of Biological Agents. In 2010 the European Commission (DG Justice, Freedom and Security) funded a 3-year project named AniBioThreat to improve the EU's capacity to counter animal bioterrorism threats. A detection portfolio with screening methods for botulism agents and incidents was needed to improve tracking and tracing of accidental and deliberate contamination of the feed and food chain with botulinum neurotoxins and other Clostridia. The complexity of this threat required acquiring new genetic information to better understand the diversity of these Clostridia and develop detection methods targeting both highly specific genetic markers of these Clostridia and the neurotoxins they are able to produce. Several European institutes participating in the AniBioThreat project collaborated on this program to achieve these objectives. Their scientific developments are discussed here.

The workshop on animal botulism in Europe.

A workshop on animal botulism was held in Uppsala, Sweden, in June 2012. Its purpose was to explore the current status of the disease in Europe by gathering the European experts in animal botulism and to raise awareness of the disease among veterinarians and others involved in biopreparedness. Animal botulism is underreported and underdiagnosed, but an increasing number of reports, as well as the information gathered from this workshop, show that it is an emerging problem in Europe. The workshop was divided into 4 sessions: animal botulism in Europe, the bacteria behind the disease, detection and diagnostics, and European collaboration and surveillance. An electronic survey was conducted before the workshop to identify the 3 most needed discussion points, which were: prevention, preparedness and outbreak response; detection and diagnostics; and European collaboration and surveillance. The main conclusions drawn from these discussions were that there is an urgent need to replace the mouse bioassay for botulinum toxin detection with an in vitro test and that there is a need for a European network to function as a reference laboratory, which could also organize a European supply of botulinum antitoxin and vaccines. The foundation of such a network was discussed, and the proposals are presented here along with the outcome of discussions and a summary of the workshop itself.