Botulinum Type Research Articles

Five-Year Retrospective Review of Cases with Benign Essential Blepharospasm and Hemifacial Spasm Presenting in a Tertiary Eye Care Center in North India

ABSTRACTRetrospective analysis of epidemiological and clinical characteristics of patients diagnosed with benign essential blepharospasm and hemifacial spasm who reported to the oculoplasty clinic of a tertiary eye care center in north India between January 2010 and April 2015 was carried out. Dry eye, as well as all the local factors that can cause blepharospasm or hemifacial spasm, was ruled out. Systemic evaluation was done to rule out any neurological disorder. A detailed history was taken to rule out any associated psychiatric disorders as well as use of any medication which could be responsible for dystonic movements. In every patient of hemifacial spasm, magnetic resonance imaging (MRI) of the brain was done for any facial nerve compression or tumor involving posterior fossa. Botulinum type A injections were given after assessing their requirements on the basis of guidelines given by Jankovic et al.

Randomised controlled safety study of a Clostridium botulinum type C vaccine for the prevention of Equine Grass Sickness: evidence of vaccine immunogenicity and safety in the horse

Randomised controlled safety study of a Clostridium botulinum type C vaccine for the prevention of Equine Grass Sickness: evidence of vaccine immunogenicity and safety in the horse

Vaccination against Equine Grass Sickness: piloting a clinical field trial of Clostridium botulinum type C toxoid in the United Kingdom

Vaccination against Equine Grass Sickness: piloting a clinical field trial of Clostridium botulinum type C toxoid in the United Kingdom

Equine grass sickness, but not botulism, causes autonomic and enteric neurodegeneration and increases soluble N-ethylmaleimide-sensitive factor attachment receptor protein expression within neuronal perikarya.

Equine grass sickness (EGS) is of unknown aetiology. Despite some evidence suggesting that it represents a toxico-infection with Clostridium botulinum types C and/or D, the effect of EGS on the functional targets of botulinum neurotoxins, namely the soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins, is unknown. Further, while it is commonly stated that, unlike EGS, equine botulism is not associated with autonomic and enteric neurodegeneration, this has not been definitively assessed. To determine: 1) whether botulism causes autonomic and enteric neurodegeneration; and 2) the effect of EGS on the expression of SNARE proteins within cranial cervical ganglion (CCG) and enteric neuronal perikarya. Descriptive study. Light microscopy was used to compare the morphology of neurons in haematoxylin-eosin stained sections of CCG and ileum from 6 EGS horses, 5 botulism horses and 6 control horses. Immunohistochemistry was used to compare the expression of synaptosomal-associated protein-25, synaptobrevin (Syb) and syntaxin within CCG neurons, and of Syb in enteric neurons, from horses with EGS, horses with botulism and control horses. The concentrations of these SNARE proteins in extracts of CCG from EGS and control horses were compared using quantitative fluorescent western blotting. EGS, but not botulism, was associated with autonomic and enteric neurodegeneration and with increased immunoreactivity for SNARE proteins within neuronal perikarya. Quantitative fluorescent western blotting confirmed increased concentrations of synaptosomal-associated protein-25, Syb and syntaxin within CCG extracts from EGS vs. control horses, with the increases in the latter 2 proteins being statistically significant. The occurrence of autonomic and enteric neurodegeneration, and increased expression of SNARE proteins within neuronal perikarya, in EGS but not botulism, suggests that EGS may not be caused by botulinum neurotoxins. Further investigation of the aetiology of EGS is therefore warranted.

Botulinum Toxin Type A Injection May Restore Ankle Strategy Use in Stroke Patients: A Preliminary Report

Introduction: Stroke causes upper motor neuron syndrome, and spasticity of foot ankle complex further leads to equine gait and posture stability disturbance. Botulinum type A (BTX-A) injection has been widely used in treating spasticity, yet few studies report its effect on posture control. This study aimed at applying BTX-A injection in treating ankle-foot complex spasticity and investigating its effect on ankle strategy use with sensory organization test. Material and method: 9 stroke patients and 5 healthy subjects were recruited. BTX-A injection was applied over calf flexor muscle group under electrical stimulation guide. Strategy score (ST) and Composite score were assessed using standard sensory organization test(SOT) with SMART Balance Master. The higher ST and Composite score indicated that the subjects used more ankle strategy. The individual test conditions were as following: EO: eyes open; EC: eyes closed; SV: swayed vision and fixed support; EOSS: eyes open and swayed support; ECSS: eyes closed and swayed support; SVSS: swayed vision and swayed support). For the patient group, SOT was performed before BTX-A injection, and at 4 weeks, 8 weeks and 12 weeks follow up. For control group, the assessments were performed once. Generalized estimating equation was implemented for data analysis, and set p<0.05 as statistical significance.

Principal component analysis and kinetic model adjustment to describe the community-level physiological profiles (CLPP) in the sediments of the river Minho estuary (Northern Portugal)

Principal component analysis and kinetic model adjustment to describe the community-level physiological profiles (CLPP) in the sediments of the river Minho estuary (Northern Portugal)

Is downer cow syndrome related to chronic botulism?

The present work was directed to investigate the relationship between Downer cow syndrome (DCS) and chronic botulism in dairy cattle. For this purpose, a total of 52 fresh calving downer cows and 206 apparently healthy cows at 14 dairy farms were investigated for Clostridium botulinum ABE and CD antibody levels, C. botulinum and botulinum neurotoxin in rumen fluids as well as in faeces. Results indicated that the downer cows had higher IgG titers for C. botulinum ABE and CD than the healthy cows. All tested rumen fluids were negative for BoNT and C. botulinum. BoNT/D, however, and C. botulinum type D spores were detected in faecal samples of healthy and downer cows in the selected farms. In conclusion, the presence of a significantly higher C. botulinum ABE and CD antibody levels in DCS cows than in the healthy animals suggests that chronic C. botulinum toxico-infection could be a predisposing factor for DCS.

Evidence for a natural humoral response in dairy cattle affected by persistent botulism sustained by non-chimeric type C strains

Bovine botulism is a sporadic acute disease that usually causes catastrophic losses in the herds. The unusual clinical evolution of a persistent mild outbreak in a dairy herd, prompted us to characterize the neurotoxin gene profile of the strain involved and to evaluate whether seroconversion had occurred.Diagnosis was based on mild classical symptoms and was supported by PCR and bacteriological findings, which revealed the involvement of a non-mosaic type C strain.An in-house ELISA was developed to detect antibodies to botulinum neurotoxin type C and its performance was evaluated in a vaccination study.Fifty days after the index case, fecal and serum samples were collected from the 14 animals of the herd and screened for Clostridium botulinum and anti-botulinum neurotoxin antibodies type C, respectively. The in-house developed ELISA was also used to test 100 sera samples randomly collected from 20 herds.Strong ELISA reactions were observed in 3 convalescent and 5 asymptomatic animals involved in the studied outbreak. The ELISA-positive cows all tested positive for non-mosaic C. botulinum type C in the feces and the same strain was also detected in the alfalfa hay, suspected to be the carrier source. Ten out of the 100 randomly collected sera tested positive for anti-botulinum neurotoxin type C antibodies: 7 had borderline values and 3 from the same herd showed titers three times higher than the cut-off.We concluded that type C botulism in cattle may occur with variable severity and that prolonged exposure to sublethal doses of botulinum neurotoxin C may occur, resulting in detectable antibodies.

An acute outbreak of equine dysautonomia (equine grass sickness) in a group of eight Przewalski's horses (Equus ferus [caballus] przewalskii)

Equine grass sickness (EGS) is a neurodegenerative disease affecting grazing equids of which a single case of the chronic clinical presentation has previously been reported in a Przewalski's horse (Equus ferus [caballus] przewalskii). A group of 8 Przewalski's horses were moved to a new enclosure, recently vacated by a group of 4 Eastern kiang (Equus kiang holdereri) that showed no evidence of disease. After 23 days the first Przewalski's horse showed clinical signs of acute EGS including flank sweating, belly kicking, rapid loss of body condition, cessation of faecal passage, nasogastric reflux and mouthing water. It was subjected to euthanasia within 48 h due to lack of therapeutic response. Within 24 h of this first case developing clinical signs, a further 5 Przewalski's horses showed similar clinical signs of acute EGS and were subjected to euthanasia. Post mortem examinations confirmed acute EGS, with all animals demonstrating typical chromatolysis, cytoplasmic hypereosinophilia, cellular swelling, vacuolation, pyknosis and loss of nuclei in approximately 90% of neurones in the cranial cervical and cranial mesenteric ganglia and myenteric and submucosal plexi of the ileum. Two Przewalski's horses within the group showed no clinical signs of disease. No single pathogen was identified as the causal agent, but the epidemiological pattern of the outbreak was typical for that previously reported for acute EGS in domestic equids. All affected animals and the 2 surviving Przewalski's horses had low antibody titres to Clostridium botulinum type C. This is the first report of acute EGS in a herd of Przewalski's horses.

The pattern of growth observed for Clostridium botulinum type A1 strain ATCC 19397 is influenced by nutritional status and quorum sensing: a modelling perspective.

Botulinum neurotoxins (BoNTs) produced by the anaerobic bacterium Clostridium botulinum are the most poisonous substances known to mankind. However, toxin regulation and signals triggering synthesis as well as the regulatory network and actors controlling toxin production are unknown. Experiments show that the neurotoxin gene is growth phase dependent for C. botulinum type A1 strain ATCC 19397, and toxin production is influenced both by culture conditions and nutritional status of the medium. Building mathematical models to describe the genetic and molecular machinery that drives the synthesis and release of BoNT requires a simultaneous description of the growth of the bacterium in culture. Here, we show four plausible modelling options which could be considered when constructing models describing the pattern of growth observed in a botulinum growth medium. Commonly used bacterial growth models are unsuitable to fit the pattern of growth observed, since they only include monotonic growth behaviour. We find that a model that includes both the nutritional status and the ability of the cells to sense their surroundings in a quorum-sensing manner is most successful at explaining the pattern of growth obtained for C. botulinum type A1 strain ATCC 19397.

Structure–function discrepancy in Clostridium botulinum C3 toxin for its rational prioritization as a subunit vaccine

Clostridium botulinum is anaerobic pathogenic bacterium causing food-born botulism in human and animals by producing botulinum neurotoxins A-H, C2, and C3 cytotoxins. Physiological group III strains (type C and D) of this bacterium are capable of producing C2 and C3 toxins in cattle and avian. Herein, we have revealed the structure–function disparity of C3 toxins from two different C. botulinum type C phage (CboC) and type D phage (CboD) to design avirulent toxins rationally. Structure–function discrepancy of the both toxins was computationally evaluated from their homology models based on the conservation in sequence–structure–function relationships upon covariation and point mutations. It has shown that 8 avirulent mutants were generated from CboC of 34 mutants while 27 avirulent mutants resulted from CboD mutants. No major changes were found in tertiary structure of these toxins; however, some structural variations appeared in the coiled and loop regions. Correlated mutation on the first residue would disorder or revolutionize the hydrogen bonding pattern of the coevolved pairs. It suggested that the residues coupling in the local structural environments were compensated with coevolved pairs so as to preserve a pseudocatalytic function in the avirulent mutants. Avirulent mutants of C3 toxins have shown a stable structure with a common blue print of folding process and also attained a near-native backrub ensemble. Thus, we concluded that selecting the site-directed mutagenesis sites are very important criteria for designing avirulent toxins, in development of rational subunit vaccines, to cattle and avian, but the vaccine specificity can be determined by the C3 toxins of C. botulinum harboring phages.

Effect of Fill Temperature on Clostridium botulinum Type A Toxin Activity during the Hot Filling of Juice Bottles

The potential threat of terrorist attacks against the United States food supply using neurotoxin produced by Clostridium botulinum (BoNT) has resulted in the need for studying the effect of various food process operations on the bioavailability of this toxin. The objective of this study was to evaluate C. botulinum type A neurotoxin bioavailability after a simulated hot fill juice bottling operation. C. botulinum type A acid mud toxin (∼10(6) mouse lethal dose [MLD50]/ml) was deposited into juice bottles at an experimentally determined fastest cooling spot. Bottles (12 or 20 oz [355 and 592 ml]) were filled with either apple juice or an orange drink, at 80 or 85°C, in either upright or inverted orientations. Toxicity of the juice was evaluated as a function of holding time (1 to 2 min) by the mouse bioassay. The fastest cooling point in the upright orientation was determined to be at a bottle's bottom rim. In the inverted orientation, the fastest cooling point was in the bottle cap region. With respect to these two points, the upright bottle cooled faster than the inverted bottle, which was reflected in a higher inactivation of BoNT in the latter. For the orange drink (pH 2.9) toxicity was reduced by 0.5 × 10(6) MLD50/ml to a nondetectable level after 1 min in all bottle sizes, orientations, and temperatures as measured by the mouse bioassay. This indicates that there was at least a 0.5 × 10(6) MLD50/ml reduction in activity. Inactivation in apple juice (pH 4.0), to the same degree as in the orange drink, was found only for the inverted orientation at 85°C. Complete inactivation in apple juice for all conditions was found at a lower added toxin level of 0.25 × 10(5) MLD50/ml. In general, bottle inversion and filling at 85°C provided complete inactivation of BoNT to the 0.5 × 10(6) MLD50/ml level. All experiments resulted in the inactivation of 2.5 × 10(4) MLD50/ml of BoNT regardless of juice type, fill temperature, or bottle orientation and size.

Occurrence of C. botulinum in healthy cattle and their environment following poultry botulism outbreaks in mixed farms

Ten cattle farms located in an area with a recent history of poultry botulism outbreaks were investigated to evaluate the occurrence of toxigenic C. botulinum in healthy cattle. Environmental samples in the 10 cattle farms and bovine fecal contents in farms with a confirmed environmental contamination were collected. Detection of C. botulinum toxin genes C, D, C/D, D/C and E was performed using real-time PCR. 4.9% (7/143) of the environmental samples collected in the 10 investigated cattle farms were positive for C. botulinum type C/D. Theses samples (boot-swabs in stalls and on pasture and water of a stream) were collected in 3 different farms. One cow dung sample and 3 out of 64 fecal contents samples collected in a single farm were also positive for C. botulinum type C/D. This study demonstrates that cattle are probably indirectly contaminated via poultry botulism in the area and that they can be intermittent carrier of C. botulinum type C/D after poultry botulism outbreaks in mixed farms.

Impact of Botulinum Neurotoxin Pyloric Injection During Laparoscopic Sleeve Gastrectomy on Postoperative Gastric Leak: a Clinical Randomized Study.

The most frequent and most feared complication after laparoscopic sleeve gastrectomy (LSG) is gastric leak (GL). We hypothesize that botulinum neurotoxin (botulinum type A (BTX-A)) injection into the pyloric sphincter muscle at the time of operation may decrease the risk of postoperative GL. Consecutive patients with morbid obesity (MO) treated by LSG were enrolled. Patients were randomly allocated into two groups: group I (intrapyloric BTX-A injection was performed) and group II (no injection was performed). The primary outcome measure was number of patients developing GL. Secondary outcome measures were percent of excess weight loss, postoperative complications, and their management. One hundred and fifteen patients (86 (74.8 %) females) were randomized into two groups of 57 patients (group I) and 58 patients (group II). Four patients in group II developed GL versus no patient in group I (P = 0.04). Ten patients in group I and two in group II developed refractory epigastric pain (P = 0.01). Other complication rates were comparable for both groups. Mean preoperative BMI of patients in both groups had significantly decreased from 54.64 ± 6.82 to 42.99 ± 5.3 at 6 months and to 39.09 ± 5.14 at 12 months (P < 0.001). LSG is an effective, safe, and minimally invasive procedure for treatment of MO. No patient in whom pyloric BTX-A injection was performed developed postoperative GL versus four patients in whom injection was not performed. The difference in GL rate was statistically significant, thus favoring the use of pyloric BTX-A injection during LSG.

Single-tube nested PCR assay for the detection of avian botulism in cecal contents of chickens

This paper describes a novel diagnostic method for the detection of avian botulism caused by Clostridium botulinum type C and C/D, using single-tube nested PCR assay. This assay was developed to overcome the disadvantages of bioassays used in experiments with mice. Three primer pairs including an antisense primer were designed to target the N-terminal of the toxin gene from C. botulinum types C and C/D. The specificity of the PCR assay was confirmed by using 33 bacterial strains and chicken cecal contents from farms that experienced botulism outbreaks. The detection limit for purified DNA was 1.1 fg/μl, and for bacterial spores was 4.3 spores/200 mg of cecal contents. While checking for specificity of the PCR assay, the reactions with the templates form C. botulinum type C and C/D which were tested became positive, but the rest of the reactions turned negative. However, the results for all clinical samples (n = 8) were positive. The PCR assay results for cecal samples obtained from 300 healthy chickens (150 Korean native chickens and 150 broilers) were all negative. This assay is rapid and straightforward and evades ethical issues associated with mouse bioassay. Moreover, it is more economical than real-time PCR.

High pressure thermal inactivation of Clostridium botulinum type E endospores - kinetic modeling and mechanistic insights.

Cold-tolerant, neurotoxigenic, endospore forming Clostridium (C.) botulinum type E belongs to the non-proteolytic physiological C. botulinum group II, is primarily associated with aquatic environments, and presents a safety risk for seafood. High pressure thermal (HPT) processing exploiting the synergistic effect of pressure and temperature can be used to inactivate bacterial endospores. We investigated the inactivation of C. botulinum type E spores by (near) isothermal HPT treatments at 300–1200 MPa at 30–75°C for 1 s to 10 min. The occurrence of heat and lysozyme susceptible spore fractions after such treatments was determined. The experimental data were modeled to obtain kinetic parameters and represented graphically by isoeffect lines. In contrast to findings for spores of other species and within the range of treatment parameters applied, zones of spore stabilization (lower inactivation than heat treatments alone), large heat susceptible (HPT-induced germinated) or lysozyme-dependently germinable (damaged coat layer) spore fractions were not detected. Inactivation followed first order kinetics. Dipicolinic acid release kinetics allowed for insights into possible inactivation mechanisms suggesting a (poorly effective) physiologic-like (similar to nutrient-induced) germination at ≤450 MPa/≤45°C and non-physiological germination at >500 MPa/>60–70°C. Results of this study support the existence of some commonalities in the HPT inactivation mechanism of C. botulinum type E spores and Bacillus spores although both organisms have significantly different HPT resistance properties. The information presented here contributes to closing the gap in knowledge regarding the HPT inactivation of spore formers relevant to food safety and may help industrial implementation of HPT processing. The markedly lower HPT resistance of C. botulinum type E spores compared with the resistance of spores from other C. botulinum types could allow for the implementation of milder processes without endangering food safety.

Patient considerations in the treatment of cervical dystonia: focus on botulinum toxin type A.

Cervical dystonia is the most common form of focal dystonia characterized by involuntary muscle contractions causing abnormal movements and posturing of the head and neck and is associated with significant pain. Botulinum toxin is considered first-line therapy in the treatment of pain and abnormal head posturing associated with cervical dystonia. There are currently three botulinum toxin type A neurotoxins and one botulinum type B neurotoxin commercially available and US Food and Drug Administration (FDA) labeled for the treatment of cervical dystonia. This review will focus on the efficacy, safety, and therapeutic use of botulinum type A neurotoxins in the treatment of cervical dystonia. We conclude with a discussion of factors influencing toxin selection including therapeutic effect, duration of effect, side effect profile, cost, and physician preference.

Spatial, Temporal, and Matrix Variability of Clostridium botulinum Type E Toxin Gene Distribution at Great Lakes Beaches.

Clostridium botulinum type E toxin is responsible for extensive mortality of birds and fish in the Great Lakes. The C. botulinum bontE gene that produces the type E toxin was amplified with quantitative PCR from 150 sloughed algal samples (primarily Cladophora species) collected during summer 2012 from 10 Great Lakes beaches in five states; concurrently, 74 sediment and 37 water samples from four sites were also analyzed. The bontE gene concentration in algae was significantly higher than in water and sediment (P < 0.05), suggesting that algal mats provide a better microenvironment for C. botulinum. The bontE gene was detected most frequently in algae at Jeorse Park and Portage Lake Front beaches (Lake Michigan) and Bay City State Recreation Area beach on Saginaw Bay (Lake Huron), where 77, 100, and 83% of these algal samples contained the bontE gene, respectively. The highest concentration of bontE was detected at Bay City (1.98 × 10(5) gene copies/ml of algae or 5.21 × 10(6) g [dry weight]). This study revealed that the bontE gene is abundant in the Great Lakes but that it has spatial, temporal, and matrix variability. Further, embayed beaches, low wave height, low wind velocity, and greater average water temperature enhance the bontE occurrence.

Detection of Clostridium botulinum in liquid manure and biogas plant wastes.

Biogas plants have been considered as a source for possible amplification and distribution of pathogenic bacteria capable of causing severe infections in humans and animals. Manure and biogas wastes could be sources for spore-forming bacteria such as Clostridium botulinum. In the present study, 24 liquid manure and 84 biogas waste samples from dairies where the majority of the cows suffered from chronic botulism were investigated for the presence of botulinum neurotoxins (BoNT) and C. botulinum spores. The prevalence of BoNT/A, B, C, D, and E in biogas wastes was 16.6, 8.3, 10.7, 7.1, and 10.8 %, respectively, while in manure, the prevalence was 0.0, 0.0, 0.0, 8.3, and 4.1 %, respectively. After enrichment of samples in reinforced cultural medium, they were tested for C. botulinum BoNT/A, B, C, D, and E using ELISA (indirect C. botulinum detection). The prevalence of C. botulinum type A, B, C, D, and E samples in biogas wastes was 20.2, 15.5, 19, 10.7, and 34.8 %, respectively, while the prevalence in liquid manure was 0.0, 0.0, 0.0, 8.3, and 12.5 %, respectively. In conclusion, the occurrence of BoNT and C. botulinum spores in biogas waste of diseased animals indicates an increased and underestimated hygienic risk. Application of digestates from biogas fermentations as fertilizers could lead to an accumulation of long lifespan spores in the environment and could be a possible health hazard.

Cost-utility analysis of botulinum toxin type A products for the treatment of cervical dystonia.

A cost-utility analysis of botulinum toxin type A products for the treatment of cervical dystonia (CD) was conducted. A cost-utility analysis of botulinum toxin type A products was conducted from the U.S. government perspective using a decision-analysis model with a one-year time horizon. Probabilities of the model were taken from several studies using the three botulinum type A products approved by the Food and Drug Administration for the treatment of CD: onabotulinumtoxinA (Botox), abobotulinumtoxinA (Dysport), and incobotulinumtoxinA (Xeomin). The main outcome measurement was successful treatment response with botulinum toxin type A, measured in quality-adjusted life years (QALYs). Response was defined as a patient who experienced improvement of CD symptoms without a severe adverse event. Probabilistic sensitivity analysis was conducted to test robustness of the base-case results. All three botulinum toxin type A agents were cost-effective at a willingness-to-pay threshold of $100,000 per QALY. Xeomin was the most cost-effective with a cost-effectiveness ratio of $27,548 per QALY. Xeomin was dominant over the alternative agents with equivalent efficacy outcomes and lower costs. Dysport had the second lowest cost-effectiveness ratio ($36,678), followed by Botox ($49,337). The probabilistic sensitivity analysis supported the results of the base-case analysis. Dysport was associated with the lowest wastage (2.2%), followed by Xeomin (10%) and Botox (22.9%). A cost-utility analysis found that Xeomin was the more cost-effective botulinum toxin type A product compared with Botox and Dysport for the treatment of CD. Wastage associated with the respective products may have a large effect on the cost-effectiveness of the agents.