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genome sequence of the group iii clostridium botulinum strain eklund-c.the neurotoxins produced by clostridium botulinum strains are among the world's most potent toxins and are the causative agents of paralytic botulism. here, we present the draft genome sequence of the group iii c. botulinum strain eklund-c, including a pseudolysogen-like bacteriophage that harbors the type c neurotoxin operon.201323516187
use of a new functional dual coating (fdc) assay to measure low toxin levels in serum and food samples following an outbreak of human botulism.clostridium botulinum type a toxin is the most prevalent cause of naturally occurring outbreaks of human botulism in the world. the active dichain neurotoxin molecule is composed of a heavy chain (h-chain) of ~100 kda with the carboxy-terminal end consisting of a receptor-binding (hc) domain, while the amino-terminal (hn) domain is linked by a critical disulfide bond to a light chain (l-chain) of ~50 kda. although the mouse bioassay (mba) is traditionally used to confirm the presence of toxin in ...201323518650
c3 exoenzyme lacks effects on peripheral axon regeneration in vivo.peripheral nerve injury triggers the activation of the small gtpase rhoa in spinal motor and peripheral sensory neurons. c3 transferase, an exoenzyme produced by clostridium botulinum that inactivates rhoa by adp-ribosylation, has been successfully applied in central nervous system (cns) lesion models to facilitate regeneration functionally and morphologically. until now it has not been demonstrated if c3bot exerts positive effects on peripheral axon regeneration as well. in organotypic spinal c ...201323521641
structural insights into the functional role of the hcn sub-domain of the receptor-binding domain of the botulinum neurotoxin mosaic serotype c/d.botulinum neurotoxin (bont), the causative agent of the deadly neuroparalytic disease botulism, is the most poisonous protein known for humans. produced by different strains of the anaerobic bacterium clostridium botulinum, bont effects cellular intoxication via a multistep mechanism executed by the three modules of the activated protein. endocytosis, the first step of cellular intoxication, is triggered by the ~50 kda, heavy-chain receptor-binding domain (hcr) that is specific for a ganglioside ...201323523511
development of a cell-based functional assay for the detection of clostridium botulinum neurotoxin types a and e.the standard procedure for definitive detection of bont-producing clostridia is a culture method combined with neurotoxin detection using a standard mouse bioassay (mba). the mouse bioassay is highly sensitive and specific, but it is expensive and time-consuming, and there are ethical concerns due to use of laboratory animals. cell-based assays provide an alternative to the mba in screening for bont-producing clostridia. here, we describe a cell-based assay utilizing a fluorescence reporter cons ...201323533420
pathogenic psychrotolerant sporeformers: an emerging challenge for low-temperature storage of minimally processed foods.sporeforming bacteria are a significant problem in the food industry as they are ubiquitous in nature and capable of resisting inactivation by heat and chemical treatments designed to inactivate them. beyond spoilage issues, psychrotolerant sporeformers are becoming increasingly recognized as a potential hazard given the ever-expanding demand for refrigerated processed foods with extended shelf-life. in these products, the sporeforming pathogens of concern are bacillus cereus, bacillus weihenste ...201323536982
two-component signal transduction system cbo0787/cbo0786 represses transcription from botulinum neurotoxin promoters in clostridium botulinum atcc 3502.blocking neurotransmission, botulinum neurotoxin is the most poisonous biological substance known to mankind. despite its infamy as the scourge of the food industry, the neurotoxin is increasingly used as a pharmaceutical to treat an expanding range of muscle disorders. whilst neurotoxin expression by the spore-forming bacterium clostridium botulinum appears tightly regulated, to date only positive regulatory elements, such as the alternative sigma factor botr, have been implicated in this contr ...201323555260
detailed analysis of metagenome datasets obtained from biogas-producing microbial communities residing in biogas reactors does not indicate the presence of putative pathogenic microorganisms.in recent years biogas plants in germany have been supposed to be involved in amplification and dissemination of pathogenic bacteria causing severe infections in humans and animals. in particular, biogas plants are discussed to contribute to the spreading of escherichia coli infections in humans or chronic botulism in cattle caused by clostridium botulinum. metagenome datasets of microbial communities from an agricultural biogas plant as well as from anaerobic lab-scale digesters operating at di ...201323557021
selective binding of 2'-f-c-di-gmp to ct-e88 and cb-e43, new class i riboswitches from clostridium tetani and clostridium botulinum respectively.c-di-gmp is a second messenger in bacteria and partly regulates bacterial physiology by binding to class i and ii riboswitches. four class i c-di-gmp riboswitch aptamer candidates, ct-e88, cb-17b, cb-e43 and cd-630 rnas, selected from a gemm rna sequence motif in the rfam database, were expressed and experimentally verified to bind to c-di-gmp. the two newly characterized c-di-gmp riboswitches, ct-e88 and cb-e43, bound c-di-gmp with nanomolar kd whereas the affinities of cb-17b and cd-630 for c- ...201323559271
plasmid-borne type e neurotoxin gene clusters in clostridium botulinum strains.a collection of 36 clostridium botulinum type e strains was examined by pulsed-field gel electrophoresis (pfge) and southern hybridization with probes targeted to bote and orfx1 in the neurotoxin gene cluster. three strains were found to contain neurotoxin subtype e1 gene clusters in large plasmids of about 146 kb in size.201323563942
alternative sigma factor sigk has a role in stress tolerance of group i clostridium botulinum strain atcc 3502.the role of the alternative sigma factor sigk in cold and osmotic stress tolerance of clostridium botulinum atcc 3502 was demonstrated by induction of sigk after temperature downshift and exposure to hyperosmotic conditions and by impaired growth of the sigk mutants under the respective conditions.201323563953
evaluation of the processing of perna perna mussels: the influence of water quality involved in the cooling operations in the physico-chemical and microbiological characteristics of the product.the state of santa catarina in brazil is a large producer of perna perna mussels. however, raw and processed mussels have a short shelf life because of their high microbiological count, such as vibrio spp. this study evaluated the microbiological and physicochemical quality of raw and ready-to-eat mussels and the quality of water and ice used in the processing of mussels.201323585097
presence of clostridium botulinum and botulinum toxin in milk and udder tissue of dairy cows with suspected botulism.clostridium botulinum is an anaerobic spore-forming bacterium prevalent in the environment, and causes botulism in man and animals via toxins. dairy cattle may be contaminated or infected by feed, water or other environmental factors. milk may also carry the pathogen. hence, milk and udder samples need to be tested. the number of clinical cases of bovine botulism in germany has been increasing since the mid-1990s. besides routine samples, additional 99 milk samples from 37 farms, and 51 udder sa ...201323585115
development and characterization of six monoclonal antibodies to hemagglutinin-70 of clostridium botulinum and their application in a sandwich elisa.botulinum neurotoxins (bont) are produced by clostridium botulinum and cause severe neuroparalytic disease that if not treated quickly is often fatal. the toxin is produced as a 150 kda precursor protein (holotoxin) that is enzymatically cleaved to form two subunits, heavy and light chains, linked by a single disulfide bond. seven toxin serotypes are known. bont serotypes a1 and b1 are secreted as precursor toxic complexes (ptc) containing of the toxin and non-toxic associated proteins (naps) co ...201323600499
genetic diversity of the flagellin genes of clostridium botulinum groups i and ii.botulinum neurotoxins (bonts) are produced by phenotypically and genetically different clostridium species, including clostridium botulinum and some strains of clostridium baratii (serotype f) and clostridium butyricum (serotype e). bont-producing clostridia responsible for human botulism encompass strains of group i (secreting proteases, producing toxin serotype a, b, or f, and growing optimally at 37°c) and group ii (nonproteolytic, producing toxin serotype e, b, or f, and growing optimally at ...201323603687
type c and c/d toxigenic clostridium botulinum is not normally present in the intestine of healthy broilers.toxigenic clostridium botulinum spores are widely distributed in wetland environments and are frequently recovered from healthy wild birds, where ingestion of toxic maggots from carcasses is considered a major initiating factor for botulism outbreaks. toxic carcasses can perpetuate an existing outbreak also in poultry, but their importance as an initiating factor for poultry botulism is less clear. although toxigenic c. botulinum can be recovered from healthy broilers during outbreaks, there are ...201323623689
purification and characterization of neurotoxin complex from a dual toxin gene containing clostridium botulinum strain ps-5.botulinum neurotoxins are produced as a toxin complex (tc) which consists of neurotoxin (nt) and neurotoxin associated proteins. the characterization of nt in its native state is an essential step for developing diagnostics and therapeutic countermeasures against botulism. the presence of nt genes was validated by pcr amplification of toxin specific fragments from genomic dna of clostridium botulinum strain ps-5 which indicated the presence of both serotype a and b genes on ps-5 genome. further, ...201323625059
[the new microbiological hazards in food].this paper describes the new microbiological hazards in food. for protecting human health, nowadays food safety authorities face with many challenges, that years ago were largely unheard. in 2011 verocytotoxigenic escherichia coli o104:h4 has been isolated in germany. strain came from fenugreek sprouts originated from egypt. it was characterized by unique features such as presence of enteroaggregative escherichia coli genes (aata, aggr, aap, agga, aggc) and resistance to most antibiotics. in pol ...201223631259
clostridium botulinum strain af84 contains three neurotoxin gene clusters: bont/a2, bont/f4 and bont/f5.sanger and shotgun sequencing of clostridium botulinum strain af84 type af and its botulinum neurotoxin gene (bont) clusters identified the presence of three bont gene clusters rather than the expected two. the three toxin gene clusters consisted of bont subtypes a2, f4 and f5. the bont/a2 and bont/f4 gene clusters were located within the chromosome (the latter in a novel location), while the bont/f5 toxin gene cluster was located within a large 246 kb plasmid. these findings are the first ident ...201323637798
environmental factors influencing the prevalence of a clostridium botulinum type c/d mosaic strain in nonpermanent mediterranean wetlands.between 1978 and 2008, 13 avian botulism outbreaks were recorded in the wetlands of mancha húmeda (central spain). these outbreaks caused the deaths of around 20,000 birds from over 50 species, including globally endangered white-headed ducks (oxyura leucoceophala). here, a significant association was found between the number of dead birds recorded in each botulism outbreak and the mean temperature in july (always >26°c). the presence of clostridium botulinum type c/d in wetland sediments was de ...201323645197
the type f6 neurotoxin gene cluster locus of group ii clostridium botulinum has evolved by successive disruption of two different ancestral precursors.genome sequences of five different group ii (nonproteolytic) clostridium botulinum type f6 strains were compared at a 50-kb locus containing the neurotoxin gene cluster. a clonal origin for these strains is indicated by the fact that sequences were identical except for strain eklund 202f, with 10 single-nucleotide polymorphisms and a 15-bp deletion. the essential topb gene encoding topoisomerase iii was found to have been split by the apparent insertion of 34.4 kb of foreign dna (in a similar ma ...201323645598
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 natur ...201323669132
genomic and physiological variability within group ii (non-proteolytic) clostridium botulinum.clostridium botulinum is a group of four physiologically and phylogenetically distinct bacteria that produce botulinum neurotoxin. while studies have characterised variability between strains of group i (proteolytic) c. botulinum, the genetic and physiological variability and relationships between strains within group ii (non-proteolytic) c. botulinum are not well understood. in this study the genome of group ii strain c. botulinum eklund 17b (nrp) was sequenced and used to construct a whole gen ...201323679073
infant botulism with prolonged faecal excretion of botulinum neurotoxin and clostridium botulinum for 7 months.in finland in april 2010, a 3-month old baby was diagnosed with type a infant botulism. he excreted botulinum neurotoxin and/or clostridium botulinum in his faeces until november 2010. five months of excretion was after clinical recovery and discharge from hospital. c. botulinum isolates recovered from the household dust in the patient's home were genetically identical to those found in the infant's stool samples. long-term faecal excretion of c. botulinum may pose a possible health risk for the ...201423688392
onabotulinum toxin a for treatment of chronic migraine with medication overuse.chronic migraine is a common and debilitating headache syndrome. botulinum neurotoxin (bont), a potent toxin produced by the anaerobic bacterium clostridium botulinum, used largely for treatment of disorders associated with increased muscle tone and hyperidrosis, has been recently used for patients suffering from chronic migraine. in this study, two groups of patients were treated with different dosages of bont a to verify the efficacy for chronic migraine. the results confirmed the efficacy of ...201323695040
genetic characteristics of toxigenic clostridia and toxin gene evolution.clostridia comprise a heterogenous group of environmental bacteria containing 15 pathogenic species, which produce the most potent toxins. the origin of toxins is still enigmatic. it is hypothesized that toxins exhibiting an enzymatic activity have derived from hydrolytic enzymes, which are abundantly secreted by these bacteria, and that pore-forming toxins have evolved from an ancestor transmembrane protein. the presence of related toxin genes in distinct clostridium species and the variability ...201323707611
infant botulism: first two confirmed cases in slovenia and literature review.in europe, infant botulism is a rare but probably under-diagnosed disease. with the intent to spread the awareness of this potentially life-threatening disease, we present a review of the literature with the emphasis on european epidemiology and a practical approach to diagnosis. we also report the first two confirmed cases of infant botulism in slovenia and describe our way to the final diagnosis in a clinical setting where all appropriate diagnostic tests and treatment options are not readily ...201323707631
two multidrug-resistant clinical isolates of bacteroides fragilis carry a novel metronidazole resistance nim gene (nimj).two multidrug-resistant bacteroides fragilis clinical isolates contain and express a novel nim gene, nimj, that is not recognized by the "universal" nim primers and can confer increased resistance to metronidazole when introduced into a susceptible strain on a multicopy plasmid. hmw615, an appendiceal isolate, contains at least two copies of nimj on its genome, while hmw616, an isolate from a patient with sepsis, contains one genomic copy of nimj. b. fragilis nimj is phylogenetically closer to p ...201323716049
foodborne botulism in canada, 1985-2005.during 1985-2005, a total of 91 laboratory-confirmed outbreaks of foodborne botulism occurred in canada; these outbreaks involved 205 cases and 11 deaths. of the outbreaks, 75 (86.2%) were caused by clostridium botulinum type e, followed by types a (7, 8.1%) and b (5, 5.7%). approximately 85% of the outbreaks occurred in alaska native communities, particularly the inuit of nunavik in northern quebec and the first nations population of the pacific coast of british columbia. these populations were ...201323735780
botulism with unusual rapid progression to complete paralysis in a child.botulism is a severe neuroparalytic illness which is difficult to diagnose accurately, especially in children. we report a child with type a botulism intoxication, with very rapid progression to coma-like consciousness and respiratory failure. careful physical examinations led to the suspicion of botulism, and electrophysiologic examinations, including electroencephalogram and repetitive nerve stimulation tests, further supported the diagnosis. hospitalization due to botulism had a great emotion ...201523755946
botulism in children: a diagnostic dilemma in developing countries.botulism is a well-known disease of the neuromuscular junction. it is a rare but curable cause of paralysis in paediatric population. in addition to classical clinical signs and symptoms, the diagnosis of botulism requires laboratory confirmation of intoxication by various biological tests. these include demonstration of botulinum toxin in serum or isolation of the clostridium botulinum from stool/gastric aspirates. however, it is not always possible to confirm intoxication due to unavailability ...201323763811
the effect of ph on growth of clostridium botulinum type a and expression of bonta and botr during different growth stages.in this study, the effects of ph on the growth, relative expressions of bonta and botr genes, and neurotoxin formation of foodborne pathogens clostridium botulinum type a were systematically studied throughout its growth stage. as in the previous reports, no c. botulinum growth was observed at extremely acidic ph. however, the effect of alkaline ph on the growth and neurotoxin production of c. botulinum was first revealed in this study. the maximum growth rate at ph 9.0 was similar to that at ot ...201323767855
regulation of toxin synthesis in clostridium botulinum and clostridium tetani.botulinum and tetanus neurotoxins are structurally and functionally related proteins that are potent inhibitors of neuroexocytosis. botulinum neurotoxin (bont) associates with non-toxic proteins (antps) to form complexes of various sizes, whereas tetanus toxin (tent) does not form any complex. the bont and antp genes are clustered in a dna segment called the botulinum locus, which has different genomic localization (chromosome, plasmid, phage) in the various clostridium botulinum types and subty ...201323769754
transport of the botulinum neurotoxin-associating protein, nontoxic nonhemagglutinin, across the rat small intestinal epithelial cell monolayer.botulinum neurotoxin (bont) associates with nontoxic nonhemagglutinin (ntnha) yielding a complex in culture. bont and ntnha have similar domain organizations, implying that they share common functions, although this remains unclear. here, we examined cell monolayer transport of serotype d ntnha in the rat intestinal epithelial cell line iec-6. ntnha and bont both bound to the cell and were transported across the cell layer. ntnha contains a qxw motif and a β-trefoil fold, both common in sugar ch ...201323809018
the two-component system cbo2306/cbo2307 is important for cold adaptation of clostridium botulinum atcc 3502.clostridium botulinum is a notorious foodborne pathogen. its ability to adapt to and grow at low temperatures is of interest for food safety. two-component systems (tcss) have been reported to be involved in cold-shock and growth at low temperatures. here we show the importance of tcs cbo2306/cbo2307 in the cold-shock response of c. botulinum atcc 3502. the relative expression levels of the cbo2306 and cbo2307 were up to 4.4-fold induced in the cold-shocked cultures but negatively regulated in t ...201323810493
comparative immunochemical characteristics of botulinum neurotoxin type a and its associated proteins.clostridium botulinum strains secrete their neurotoxins (bont) along with a group of neurotoxin-associated proteins (naps) that enhance the oral toxicity and provide protection to the neurotoxin against acidity, temperature and proteases in the g.i. tract. a major component of naps is hn-33, a 33 kda protein, which is also protease resistant and strongly protects bont. the complex form of bont/a is used as a commercial therapeutic formulation against many neuromuscular disorders and for cosmetic ...201323811077
evaluation of cp chromo select agar for the enumeration of clostridium perfringens from water.the european directive on drinking water quality has included mcp agar as the reference method for recovering clostridium perfringens from drinking waters. in the present study, three media (mcp, tscf and cp chromo select agar) were evaluated for recovery of c. perfringens in different surface water samples. out of 139 water samples, using a membrane filtration technique, 131 samples (94.2%) were found to be presumptively positive for c. perfringens in at least one of the culture media. green co ...201323816139
application of high-density dna resequencing microarray for detection and characterization of botulinum neurotoxin-producing clostridia.clostridium botulinum and related clostridia express extremely potent toxins known as botulinum neurotoxins (bonts) that cause severe, potentially lethal intoxications in humans. these bont-producing bacteria are categorized in seven major toxinotypes (a through g) and several subtypes. the high diversity in nucleotide sequence and genetic organization of the gene cluster encoding the bont components poses a great challenge for the screening and characterization of bont-producing strains.201323818983
distinctive g protein-dependent signaling by protease-activated receptor 2 (par2) in smooth muscle: feedback inhibition of rhoa by camp-independent pka.we examined expression of protease-activated receptors 2 (par2) and characterized their signaling pathways in rabbit gastric muscle cells. the par2 activating peptide sligrl (par2-ap) stimulated gq, g13, gi1, pi hydrolysis, and rho kinase activity, and inhibited camp formation. stimulation of pi hydrolysis was partly inhibited in cells expressing par2 sirna, gaq or gai minigene and in cells treated with pertussis toxin, and augmented by expression of dominant negative regulator of g protein sign ...201323825105
efficacy of clostridium botulinum types c and d toxoid vaccination in danish cows.in the present study the efficacy botulism vaccine (formalinised aluminum hydroxide gel adsorbed toxoid of clostridium botulinum types c and d) was evaluated in four danish dairy cows under field conditions. other four dairy herds were unvaccinated. blood serum of all animals was analyzed for specific c. botulinum types a, b, c, d and e antibodies using a developed elisa. feces of all animals were analyzed for botulinum neurotoxins (bonts) and c. botulinum spores. c. botulinum types c and d anti ...201323831724
detection limit of clostridium botulinum spores in dried mushroom samples sourced from china.a survey of dried mushrooms (lentinula edodes (shiitake) and auricularia auricula (wood ear)) sourced from china was carried out to determine the natural contamination of these mushrooms with spores of proteolytic clostridium botulinum and non-proteolytic c. botulinum. the mushrooms were collected from supermarkets and retailers in 21 cities in china during october 2008. spore loads of c. botulinum in mushrooms have a degree of uncertainty and variability and this study contributes valuable data ...201323838282
infantile botulism: a case report and review.infantile botulism is the result of ingestion of clostridium botulinum spores, and is the most common form of infection with botulism in the united states. ninety percent of cases occur in infants <6 months old. the infants typically present with vague symptoms such as hypotonia and poor feeding. this article reports an infant with confirmed infantile botulism that presented to the emergency department (ed) with complaints of decreased feeding and absence of bowel movements for >1 week.201323871478
an electrochemiluminescence assay for the detection of bio threat agents in selected food matrices and in the screening of clostridium botulinum outbreak strains associated with type a botulism.specific screening methods for complex food matrices are needed that enable unambiguous and sensitive detection of bio threat agents (btas) such as bacillus anthracis spores and microbial toxins (e.g. staphylococcal enterotoxin b (seb) and clostridial botulinum neurotoxins (bonts)). the present study describes an image-based 96-well meso scale discovery (msd) electrochemiluminescence (ecl) assay for simultaneous detection of btas in dairy milk products.201423873138
botulinum toxin complex increases paracellular permeability in intestinal epithelial cells via activation of p38 mitogen-activated protein kinase.clostridium botulinum produces a large toxin complex (l-tc) that increases paracellular permeability in intestinal epithelial cells by a mechanism that remains unclear. here, we show that mitogen-activated protein kinases (mapks) are involved in this permeability increase. paracellular permeability was measured by fitc-dextran flux through a monolayer of rat intestinal epithelial iec-6 cells, and mapk activation was estimated from western blots. l-tc of c. botulinum serotype d strain 4947 increa ...201323884081
combined high pressure and thermal processing on inactivation of type a and proteolytic type b spores of clostridium botulinum.the aim of this study was to determine the resistance of multiple strains of clostridium botulinum type a and proteolytic type b spores exposed to combined high pressure and thermal processing and compare their resistance with clostridium sporogenes pa3679 and bacillus amyloliquefaciens tmw-2.479-fad-82 spores. the resistance of spores suspended in n-(2acetamido)-2-aminoethanesulfonic acid (aces) buffer (0.05 m, ph 7.0) was determined at a process temperature of 105°c, with high pressures of 600 ...201323905794
sugar-induced conformational change found in the ha-33/ha-17 trimer of the botulinum toxin complex.large-sized botulinum toxin complex (l-tc) is formed by conjugation of neurotoxin, nontoxic nonhemagglutinin and hemagglutinin (ha) complex. the ha complex is formed by association of three ha-70 molecules and three ha-33/ha-17 trimers, comprised of a single ha-17 and two ha-33 proteins. the ha-33/ha-17 trimer isolated from serotype d l-tc has the ability to bind to and penetrate through the intestinal epithelial cell monolayer in a sialic acid-dependent manner, and thus it plays an important ro ...201323916708
characterization of botulinum neurotoxin a subtypes 1 through 5 by investigation of activities in mice, in neuronal cell cultures, and in vitro.botulinum neurotoxins (bonts) are synthesized by clostridium botulinum and exist as seven immunologically distinct serotypes designated a through g. for most serotypes, several subtypes have now been described based on nominal differences in the amino acid sequences. bont/a1 is the most well-characterized subtype of the bont/a serotype, and many of its properties, including its potency, its prevalence as a food poison, and its utility as a pharmaceutical, have been thoroughly studied. in contras ...201323918782
microbial contents of vacuum cleaner bag dust and emitted bioaerosols and their implications for human exposure indoors.vacuum cleaners can release large concentrations of particles, both in their exhaust air and from resuspension of settled dust. however, the size, variability, and microbial diversity of these emissions are unknown, despite evidence to suggest they may contribute to allergic responses and infection transmission indoors. this study aimed to evaluate bioaerosol emission from various vacuum cleaners. we sampled the air in an experimental flow tunnel where vacuum cleaners were run, and their airborn ...201323934489
production and evaluation of a recombinant chimeric vaccine against clostridium botulinum neurotoxin types c and d.bovine botulism is a fatal disease that is caused by botulinum neurotoxins (bonts) produced by clostridium botulinum serotypes c and d and that causes great economic losses, with nearly 100% lethality during outbreaks. it has also been considered a potential source of human food-borne illness in many countries. vaccination has been reported to be the most effective way to control bovine botulism. however, the commercially available toxoid-based vaccines are difficult and hazardous to produce. ne ...201323936080
rapid oligonucleotide suspension array-based multiplex detection of bacterial pathogens.a gene-specific microsphere suspension array coupled with 15-plex polymerase chain reaction (pcr) was developed to screen bacterial samples rapidly for 10 strains of bacteria: shigella spp. (s. flexneri, s. dysenteriae, and s. sonnei), staphylococcus aureus, vibrio cholerae (serology o1 and o139), legionella pneumophila, and clostridium botulinum (types a, b, and e). fifteen sets of highly validated primers were chosen to amplify target genes simultaneously. corresponding oligonucleotide probes ...201323947819
bioluminescence assay for the highly sensitive detection of botulinum neurotoxin a activity.this article describes a novel bioluminescence assay for detecting the proteolytic activity of botulinum neurotoxins (bont) in complex matrices. the assay is capable of detecting traces of bont in blood samples as well as in food drinks. the assay was responsive to bont/a subtypes 1 to 5, and serotype e3 in buffered solutions. it was responsive to filtered clostridium botulinum supernatants and bont/a1 in complex with neurotoxin associated proteins in bouillon and milk (3.8% fat) down to 400 fm ...201323971072
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, freedo ...201323971804
management of animal botulism outbreaks: from clinical suspicion to practical countermeasures to prevent or minimize outbreaks.botulism is a severe neuroparalytic disease that affects humans, all warm-blooded animals, and some fishes. the disease is caused by exposure to toxins produced by clostridium botulinum and other botulinum toxin-producing clostridia. botulism in animals represents a severe environmental and economic concern because of its high mortality rate. moreover, meat or other products from affected animals entering the food chain may result in a public health problem. to this end, early diagnosis is cruci ...201323971806
evaluation of dna extraction methods suitable for pcr-based detection and genotyping of clostridium botulinum.sufficient quality and quantity of extracted dna is critical to detecting and performing genotyping of clostridium botulinum by means of pcr-based methods. an ideal extraction method has to optimize dna yield, minimize dna degradation, allow multiple samples to be extracted, and be efficient in terms of cost, time, labor, and supplies. eleven botulinum toxin-producing clostridia strains and 25 samples (10 food, 13 clinical, and 2 environmental samples) naturally contaminated with botulinum toxin ...201323971807
multiplex real-time pcr for detecting and typing clostridium botulinum group iii organisms and their mosaic variants.botulism is a neuroparalytic disease that can occur in all warm-blooded animals, birds, and fishes. the disease in animals is mainly caused by toxins produced by clostridium botulinum strains belonging to group iii, although outbreaks due to toxins produced by group i and ii organisms have been recognized. group iii strains are capable of producing botulinum toxins of type c, d, and c/d and d/c mosaic variants. definitive diagnosis of animal botulism is made by combining clinical findings with l ...201323971808
mass spectrometric detection of protein-based toxins.this review focuses on mass spectrometric detection of protein-based toxins, which are among the most toxic substances known. special emphasis is given to the bacterial toxins botulinum neurotoxin from clostridium botulinum and anthrax toxins from bacillus anthracis as well as the plant toxin ricin produced by ricinus communis. a common feature, apart from their extreme toxicity, is that they are composed of 2 polypeptide chains, one of which is responsible for cell uptake and another that has e ...201323971809
expression and purification of neurotoxin-associated protein ha-33/a from clostridium botulinum and evaluation of its antigenicity.botulinum neurotoxin (bont) complexes consist of neurotoxin and neurotoxin-associated proteins. hemagglutinin-33 (ha-33) is a member of bont type a (bont/a) complex. considering the protective role of ha-33 in preservation of bont/a in gastrointestinal harsh conditions and also its adjuvant role, recombinant production of this protein is favorable. thus in this study, ha-33 was expressed and purified, and subsequently its antigenicity in mice was studied.201323999711
cyclodextrin derivatives as anti-infectives.cyclodextrin derivatives can be utilized as anti-infectives with pore-forming proteins as the targets. the highly efficient selection of potent inhibitors was achieved because per-substituted cyclodextrins have the same symmetry as the target pores. inhibitors of several bacterial toxins produced by bacillus anthracis, staphylococcus aureus, clostridium perfringens, clostridium botulinum, and clostridium difficile were identified from a library of ∼200 cd derivatives. it was demonstrated that mu ...201324011515
botulinum toxin for masseter hypertrophy.benign masseter muscle hypertrophy is an uncommon clinical phenomenon of uncertain aetiology which is characterised by a soft swelling near the angle of the mandible. the swelling may on occasion be associated with facial pain and can be prominent enough to be considered cosmetically disfiguring. varying degrees of success have been reported for some of the treatment options for masseter hypertrophy, which range from simple pharmacotherapy to more invasive surgical reduction. injection of botuli ...201324018587
a cell-permeable fusion protein based on clostridium botulinum c2 toxin for delivery of p53 tumorsuppressor into cancer cells.genetically engineered bacterial protein toxins are attractive systems for delivery of exogenous proteins into the cytosol of mammalian cells. the binary c2 toxin from c. botulinum has emerged as powerful delivery vehicle, which rests on its binding/translocation component c2iia and the genetically modified adaptor domain c2in that act in concert to trigger cell uptake. the p53 tumor suppressor protein has a crucial function in suppressing carcinogenesis and is frequently inactivated by diverse ...201324039769
structure of a clostridium botulinum c143s thiaminase i/thiamin complex reveals active site architecture .thiaminases are responsible for the degradation of thiamin and its metabolites. two classes of thiaminases have been identified based on their three-dimensional structures and their requirements for a nucleophilic second substrate. although the reactions of several thiaminases have been characterized, the physiological role of thiamin degradation is not fully understood. we have determined the three-dimensional x-ray structure of an inactive c143s mutant of clostridium botulinum (cb) thiaminase ...201324079939
an atypical clostridium strain related to the clostridium botulinum group iii strain isolated from a human blood culture.a nontoxigenic strain isolated from a fatal human case of bacterial sepsis was identified as a clostridium strain from clostridium botulinum group iii, based on the phenotypic characters and 16s rrna gene sequence, and was found to be related to the mosaic c. botulinum d/c strain according to a multilocus sequence analysis of 5 housekeeping genes.201424088855
comparison of the catalytic properties of the botulinum neurotoxin subtypes a1 and a5.clostridium botulinum neurotoxins (bonts) cause the life-threatening disease botulism through the inhibition of neurotransmitter release by cleaving essential snare proteins. there are seven serologically distinctive types of bonts and many subtypes within a serotype have been identified. bont/a5 is a recently discovered subtype of type a botulinum neurotoxin which possesses a very high degree of sequence similarity and identity to the well-studied a1 subtype. in the present study, we examined t ...201324096023
prevention of rhoa activation and cofilin-mediated actin polymerization mediates the antihypertrophic effect of adenosine receptor agonists in angiotensin ii- and endothelin-1-treated cardiomyocytes.adenosine receptor activation has been shown to be associated with diminution of cardiac hypertrophy and it has been suggested that endogenously produced adenosine may serve to blunt pro-hypertrophic processes. in the present study, we determined the effects of two pro-hypertrophic stimuli, angiotensin ii (ang ii, 100 nm) and endothelin-1 (et-1, 10 nm) on ras homolog gene family, member a (rhoa)/rho-associated, coiled-coil containing protein kinase (rock) activation in cultured neonatal rat vent ...201424096734
novel clostridium botulinum toxin and dual use research of concern issues. 201424106293
botulinum neurotoxins: more and more diverse and fascinating toxic proteins. 201424106294
molecular characterization of a novel botulinum neurotoxin type h gene.we sequenced the 2 botulinum toxin gene clusters of clostridium botulinum strain ibca10-7060 type bh. the sequence of bont/h differed substantially from the sequences of the 7 known bont genes for toxin types a-g. the 5' one-third terminus of bont/h that codes for the botulinum toxin light chain differed markedly from the light chain coding sequences of toxin types a-g. the 3' two-thirds terminus of bont/h that codes for the botulinum toxin heavy chain contained a novel hn translocation domain c ...201424106295
a novel strain of clostridium botulinum that produces type b and type h botulinum toxins.clostridium botulinum strain ibca10-7060, isolated from a patient with infant botulism, produced botulinum neurotoxin type b (bont/b) and another bont that, by use of the standard mouse bioassay, could not be neutralized by any of the centers for disease control and prevention-provided monovalent polyclonal botulinum antitoxins raised against bont types a-g.201424106296
"inconvenient truths" in the pursuit of scientific knowledge and public health. 201424106297
occurrence of zoonotic clostridia and yersinia in healthy cattle.zoonotic pathogens are a frequent cause of disease worldwide. this study was designed to determine the occurrence of clostridium difficile, clostridium botulinum, and yersinia enterocolitica in cattle in southern bavaria, germany. the study population included 49 farms; 34 were dairy farms (30 also fattening beef cattle) and 15 were solely beef cattle farms. fecal and dust samples were collected from summer 2011 to summer 2012 and analyzed using a combination of enrichment procedures and real-ti ...201324112568
structure of a bimodular botulinum neurotoxin complex provides insights into its oral toxicity.botulinum neurotoxins (bonts) are produced by clostridium botulinum and cause the fatal disease botulism, a flaccid paralysis of the muscle. bonts are released together with several auxiliary proteins as progenitor toxin complexes (ptcs) to become highly potent oral poisons. here, we report the structure of a ∼760 kda 14-subunit large ptc of serotype a (l-ptc/a) and reveal insight into its absorption mechanism. using a combination of x-ray crystallography, electron microscopy, and functional stu ...201324130488
detection of anthrax and other pathogens using a unique liquid array technology.a bead-based liquid hybridization assay, luminex(®) 100™, was used to identify four pathogenic bacteria, bacillus anthracis, clostridium botulinum, francisella tularensis subsp. tularensis, and yersinia pestis, and several close relatives. hybridization between pcr-amplified target sequences and probe sequences (located within the 23s ribosomal rna gene rrl and the genes related to the toxicity of each bacterium) was detected in single-probe or multiple-probe assays, depending on the organism. t ...201424147813
ph responsive janus-like supramolecular fusion proteins for functional protein delivery.a facile, noncovalent solid-phase immobilization platform is described to assemble janus-like supramolecular fusion proteins that are responsive to external stimuli. a chemically postmodified transporter protein, dhsa, is fused with (imino)biotinylated cargo proteins via an avidin adaptor with a high degree of spatial control. notably, the derived heterofusion proteins are able to cross cellular membranes, dissociate at acidic ph due to the iminobiotin linker and preserve the enzymatic activity ...201324156787
the cold-induced two-component system cbo0366/cbo0365 regulates metabolic pathways with novel roles in group i clostridium botulinum atcc 3502 cold tolerance.the two-component system cbo0366/cbo0365 was recently demonstrated to have a role in cold tolerance of group i clostridium botulinum atcc 3502. the mechanisms under its control, ultimately resulting in increased sensitivity to low temperature, are unknown. a transcriptomic analysis with dna microarrays was performed to identify the differences in global gene expression patterns of the wild-type atcc 3502 and a derivative mutant with insertionally inactivated cbo0365 at 37 and 15°c. altogether, 1 ...201424162575
crystal structure of clostridium botulinum whole hemagglutinin reveals a huge triskelion-shaped molecular complex.clostridium botulinum ha is a component of the large botulinum neurotoxin complex and is critical for its oral toxicity. ha plays multiple roles in toxin penetration in the gastrointestinal tract, including protection from the digestive environment, binding to the intestinal mucosal surface, and disruption of the epithelial barrier. at least two properties of ha contribute to these roles: the sugar-binding activity and the barrier-disrupting activity that depends on e-cadherin binding of ha. ha ...201324165130
long lasting dysautonomia due to botulinum toxin b poisoning: clinical-laboratory follow up and difficulties in initial diagnosis.botulism is an acute form of poisoning caused by one of four types (a, b, e, f) toxins produced by clostridium botulinum, ananaerobic, spore forming bacillus. usually diagnosis of botulism is considered in patients with predominant motor symptoms: muscle weakness with intact sensation and preserved mental function.201324172031
the clo3403/clo3404 two-component system of clostridium botulinum e1 beluga is important for cold shock response and growth at low temperatures.in order to survive a temperature downshift, bacteria have to sense the changing environment and adjust their metabolism and structure. two-component signal transduction systems (tcss) play a central role in sensing and responding to many different environmental stimuli. although the nonproteolytic (group ii) clostridium botulinum represents a major hazard in chilled foods, the cold adaption mechanisms of group ii c. botulinum organisms are not known. here, we show that the clo3403/clo3404 tcs o ...201424185852
rice hulls as a possible source of clostridium botulinum type c spores for poultry. 201324186999
gene expression profiling of clostridium botulinum under heat shock stress.during growth, c. botulinum is always exposed to different environmental changes, such as temperature increase, nutrient deprivation, and ph change; however, its corresponding global transcriptional profile is uncharacterized. this study is the first description of the genome-wide gene expression profile of c. botulinum in response to heat shock stress. under heat stress (temperature shift from 37°c to 45°c over a period of 15 min), 176 c. botulinum atcc 3502 genes were differentially expressed. ...201324195079
piezoelectric ceramic (pzt) modulates axonal guidance growth of rat cortical neurons via rhoa, rac1, and cdc42 pathways.electrical stimulation is critical for axonal connection, which can stimulate axonal migration and deformation to promote axonal growth in the nervous system. netrin-1, an axonal guidance cue, can also promote axonal guidance growth, but the molecular mechanism of axonal guidance growth under indirect electric stimulation is still unknown. we investigated the molecular mechanism of axonal guidance growth under piezoelectric ceramic lead zirconate titanate (pzt) stimulation in the primary culture ...201424203571
identification of botulinum toxin type in clinical samples and foods in iran.botulism is a serious neuroparalytic disease caused by toxins of clostridium botulinum. botulinum toxin is produced under anaerobic conditions and is one of the most dangerous toxin in the world. rapid diagnosis of botulism is very essential for successful therapy. in this study, we reviewed data of cases of botulism in iran from april 2004 through march 2010.201324206405
inhibition of clostridium perfringens growth by potassium lactate during an extended cooling of cooked uncured ground turkey breasts.the u.s. department of agriculture's food safety and inspection service compliance guideline known as appendix b specifies chilling time and temperature limits for cured and uncured meat products to inhibit growth of spore-forming bacteria, particularly clostridium perfringens. sodium lactate and potassium lactate inhibit toxigenic growth of clostridium botulinum, and inhibition of c. perfringens has been reported. in this study, a cocktail of spores of three c. perfringens strains (atcc 13124, ...201324215704
analysis of the genetic distribution among members of clostridium botulinum group i using a novel multilocus sequence typing (mlst) assay.clostridium botulinum is the etiological agent of botulism. due to food-borne poisoning and the potential use of the extremely toxic botulinum neurotoxin (bont) from c. botulinum in bioterror or biocrime related actions, reliable high resolution typing methods for discriminating c. botulinum strains are needed. partial sequencing of the adk, atph, gyrb, proc, rpod and spo0a genes from 51 various c. botulinum/sporogenes isolates was performed, resulting in 37 different sequence types (sts). analy ...201424246230
quantitative real-time pcr for detection of neurotoxin genes of clostridium botulinum types a, b and c in equine samples.botulism in horses in the usa is attributed to clostridium botulinum types a, b or c. in this study, a duplex quantitative real-time pcr (qpcr) for detection of the neurotoxin genes of c. botulinum types a and b, and a singleplex qpcr for detection of the neurotoxin gene of c. botulinum type c, were optimized and validated for equine gastrointestinal, faecal and feed samples. the performance of these assays was evaluated and compared to the standard mouse bioassay (mba) using 148 well-characteri ...201424252222
vaccination of cattle with a recombinant bivalent toxoid against botulism serotypes c and d.cattle botulism is a fatal intoxication caused by botulinum neurotoxins (bonts) produced by clostridium botulinum serotypes c and d resulting in economic losses. vaccination is the most effective way to control botulism. however, the commercially available vaccines are difficult and hazardous to produce. neutralizing antibodies against the c-terminal fragment of the bont heavy chain (hc) are known to protect against lethal doses of bonts. we report the vaccination of cattle with a previously tes ...201424252701
a monoclonal antibody based capture elisa for botulinum neurotoxin serotype b: toxin detection in food.botulism is a serious foodborne neuroparalytic disease, caused by botulinum neurotoxin (bont), produced by the anaerobic bacterium clostridium botulinum. seven toxin serotypes (a-h) have been described. the majority of human cases of botulism are caused by serotypes a and b followed by e and f. we report here a group of serotype b specific monoclonal antibodies (mabs) capable of binding toxin under physiological conditions. thus, they serve as capture antibodies for a sandwich (capture) elisa. t ...201324253240
a quantitative bifunctional in vitro potency assay for botulinum neurotoxin serotype a.botulinum neurotoxin type a (bonta) is one of seven serotypes produced by clostridium botulinum (types a thru g) and is the serotype most widely used to treat both cosmetic and medical conditions. potency for botulinum toxin preparations is expressed in mouse ld50 units. there is a need to develop a non-animal based replacement for this potency assay.201424333955
effect of clostridium botulinum toxin type a injections into the deep digital flexor muscle on the range of motion of the metacarpus and carpus, and the force distribution underneath the hooves, of sound horses at the walk.in the treatment of laminitis, reducing deep digital flexor muscle (ddfm) activity might diminish its pull on the distal phalanx, thereby preventing displacement and providing pain relief. injection of clostridium botulinum toxin type a into the ddfm of horses is potentially therapeutic. however, the effects of c. botulinum toxin type a on the gait characteristics of sound horses at the walk are not known. the aim of this study was to test if a reduced ddfm activity would lead to (1) alterations ...201324360731
the effect of clostridium botulinum toxin type a injections on motor unit activity of the deep digital flexor muscle in healthy sound royal dutch sport horses.therapeutic reduction of the activity of the deep digital flexor (ddf) muscle may play a role in treatment of laminitic horses. clostridium botulinum toxin type a induces reduced muscle activity and has a spasmolytic effect in horses. in this study, the effectiveness of 200 iu c. botulinum toxin type a on reduction of ddf muscle activity was measured in seven healthy, sound, adult royal dutch sport horses. c. botulinum toxin type a was injected using ultrasound and electromyographic (emg) guidan ...201324360760
comparison of assembled clostridium botulinum a1 genomes revealed their evolutionary relationship.clostridium botulinum encompasses bacteria that produce at least one of the seven serotypes of botulinum neurotoxin (bont/a-g). the availability of genome sequences of four closely related type a1 or a1(b) strains, as well as the a1-specific microarray, allowed the analysis of their genomic organizations and evolutionary relationship. the four genomes share >90% core genes and >96% functional groups. phylogenetic analysis based on cog shows closer relations of the a1(b) strain, nctc 2916, to b1 ...201424369123
c3 rho-inhibitor for targeted pharmacological manipulation of osteoclast-like cells.the c3 toxins from clostridium botulinum (c3bot) and clostridium limosum (c3lim) as well as c3-derived fusion proteins are selectively taken up into the cytosol of monocytes/macrophages where the c3-catalyzed adp-ribosylation of rho results in inhibition of rho-signalling and characteristic morphological changes. since the fusion toxin c2in-c3lim was efficiently taken up into and inhibited proliferation of murine macrophage-like raw 264.7 cells, its effects on raw 264.7-derived osteoclasts were ...201324386487
evaluation of normalization reference genes for rt-qpcr analysis of spo0a and four sporulation sigma factor genes in clostridium botulinum group i strain atcc 3502.heat-resistant spores of clostridium botulinum can withstand the pasteurization processes in modern food processing. this poses a risk to food safety as spores may germinate into botulinum neurotoxin-producing vegetative cells. sporulation in bacillus subtilis, the model organism for sporulation, is regulated by the transcription factor spo0a and four alternative sigma factors, sigf, sige, sigg, and sigk. while the corresponding regulators are found in available genomes of c. botulinum, little i ...201424389585
inhibitions of the translocation pore of clostridium botulinum c2 toxin by tailored azolopyridinium salts protects human cells from intoxication.c2 toxin from clostridium botulinum represents the prototype of clostridial binary actin adp-ribosylating toxins which destroy the actin-cytoskeleton of mammalian cells and cause severe enteric diseases in humans and animals. after receptor-mediated endocytosis of the c2 toxin complex, the binding/translocation component c2iia forms a heptameric transmembrane pore in membranes of acidified endosomal vesicles. the separate adp-ribosyltransferase component c2i translocates through this c2iia-pore ...201424394545
the same clade of clostridium botulinum strains is causing avian botulism in southern and northern europe.avian botulism is a paralytic disease caused by clostridium botulinum-produced botulinum neurotoxins (bonts), most commonly of type c/d. it is a serious disease of waterbirds and poultry flocks in many countries in europe. the objective of this study was to compare the genetic relatedness of avian c. botulinum strains isolated in spain with strains isolated in sweden using pulsed-field gel electrophoresis (pfge). fifteen strains were isolated from spanish waterbirds using an immunomagnetic separ ...201424418766
purification, crystallization and preliminary x-ray analysis of an ha17-ha70 (ha2-ha3) complex from clostridium botulinum type c progenitor toxin.the haemagglutinin (ha) complex of clostridium botulinum type c toxin is composed of three types of subcomponents: ha33, ha17 and ha70 (also known as ha1, ha2 and ha3, respectively). here, a 260 kda ha17-ha70 complex was crystallized. his-tagged ha17 and maltose-binding-protein-tagged ha70 were expressed in escherichia coli and their complex was affinity-purified using a combination of amylose resin chromatography and nickel-nitrilotriacetic acid agarose chromatography. diffraction data were col ...201424419620
rapid identification of clostridium botulinum colonies by in vitro toxicity and antimicrobial susceptibility testing.identification of clostridium botulinum is usually based on toxin detection of broth culture by mouse bioassay and requires 7 to 10 days to complete. here, we describe an alternative in vitro procedure for direct identification of c. botulinum (types a and b) colonies which can be completed in 48 h. the method is based on toxigenicity of colonies demonstrable by enzyme immunoassay and resistance of c. botulinum to antimicrobial agents, sulpha-methoxazole, trimethoprim and cycloserine.199424420880
infant botulism in costa rica: first report from central america.clostridium botulinum is known to cause descending paralysis in infants throughout the world.201424423723
meta-analysis of d-values of proteolytic clostridium botulinum and its surrogate strain clostridium sporogenes pa 3679.foodborne botulism is a serious disease resulting from ingestion of preformed clostridium botulinum neurotoxin in foodstuff. since the 19th century, the heat resistance of this spore forming bacteria has been extensively studied in order to guarantee the public health associated with low acidic, ambient stable products. the most largely used heat resistance parameters in thermal settings of such products are the d121.1°c values (time required to have a 10-fold decrease of the spore count, at 121 ...201424448274
draft genome sequence of clostridium botulinum type b strain osaka05, isolated from an infant patient with botulism in japan.clostridium botulinum strain osaka05, which has been isolated from an infant patient with botulism in japan, is the first strain producing botulinum neurotoxin subtype b6. here, we report the draft genome sequence of c. botulinum osaka05.201424459252
a case of infant botulism infection due to consumption of untreated well-water.a 7-week-old boy with flaccid paralysis was diagnosed with infant botulism caused by clostridium botulinum toxin type a. in this case of infant botulism, untreated well-water was identified as a potential source of this infection.201424461790
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