Phylogenetic positions of Clostridium chauvoei and Clostridium septicum based on 16S rRNA gene sequences

The sequences of the 16S rRNA genes (rrs genes) of Clostridium chauvoei, the causative agent of blackleg in cattle, and the phenotypically related organism Clostridium septicum were determined. After amplification of 1,507-bp PCR fragments from the corresponding rrs genes, the sequences were determined in a single round of sequencing by using conserved region primers. A sequence similarity analysis of the sequences revealed the close phylogenetic relationship of C. chauvoei and C. septicum in Clostridium cluster I (M. D. Collins, P. A. Lawson, A. Willems, J. J. Cordoba, J. Fernandez-Garayzabal, P. Garcia, J. Cai, H. Hippe, and J. A. E. Farrow, Int. J. Syst. Bacteriol. 44:812-826, 1994), which includes Clostridium carnis, Clostridium perfringens, Clostridium botulinum, and Clostridium tetani. We found that 99.3% of the nucleotides in the genes of C. chauvoei and C. septicum are identical.

Equine botulism and acute pasture myodystrophy: new soil-borne emerging diseases in Switzerland?

In Switzerland, the incidence of equine botulism and acute pasture myodystrophy have remarkably increased in the last five years. Equine fodder-borne botulism in Europe is most likely caused by Clostridium botulinum types C and D that produce the toxins BoNT/C and BoNT/D. Horses showing signs suggestive of botulism (muscle weakness and tremors, reduced tongue tone, slow chewing, salivation and difficulties swallowing, drooping eyelids, mydriasis), especially patients that have fed on suspect fodder (mostly haylage), must be treated with anti-serum as soon as possible.They also need intensive care, which is often difficult to provide and always expensive in the face of a guarded to poor prognosis. Therefore, prevention (high standards of forage quality and vaccination) is all the more important. Pasture myodystrophy is an acute disease with signs of rhabdomyolysis and lethality rate over 90%. It affects grazing horses under frosty, windy and rainy conditions. Preliminary results indicate that Clostridium sordellii and Clostridium bifermentans producing lethal toxin may play a role in pasture myodystrophy. Our efforts concentrate on developing a new subunit vaccine for equine botulism and understanding the ethiology and pathogenesis of pasture myodystrophy with the goal of improving prevention against these highly fatal diseases that present a significant risk to our horse population.

The updated European Consensus 2009 on the use of Botulinum toxin for children with cerebral palsy

An interdisciplinary European group of clinical experts in the field of movement disorders and experienced Botulinum toxin users has updated the consensus for the use of Botulinum toxin in the treatment of children with cerebral palsy (CP). A problem-orientated approach was used focussing on both published and practice-based evidence. In part I of the consensus the authors have tabulated the supporting evidence to produce a concise but comprehensive information base, pooling data and experience from 36 institutions in 9 European countries which involves more than 10,000 patients and over 45,000 treatment sessions during a period of more than 280 treatment years. In part II of the consensus the Gross Motor Function Measure (GMFM) and Gross Motor Function Classification System (GMFCS) based Motor Development Curves have been expanded to provide a graphical framework on how to treat the motor disorders in children with CP. This graph is named "CP(Graph) Treatment Modalities - Gross Motor Function" and is intended to facilitate communication between parents, therapists and medical doctors concerning (1) achievable motor function, (2) realistic goal-setting and (3) treatment perspectives for children with CP. The updated European consensus 2009 summarises the current understanding regarding an integrated, multidisciplinary treatment approach using Botulinum toxin for the treatment of children with CP.

In the human urothelium and suburothelium, intradetrusor botulinum neurotoxin type A does not induce apoptosis: preliminary results

Intradetrusor injections of botulinum neurotoxin type A (BoNTA) are emerging as the preferred second-line treatment for neurogenic and idiopathic overactive bladder (OAB). In animal experiments, intradetrusor BoNTA injections have been shown to cause apoptosis in the bladder urothelium and suburothelium but not the detrusor.

Complete continence after botulinum neurotoxin type A injections for refractory idiopathic detrusor overactivity incontinence: patient-reported outcome at 4 weeks

Objective improvement following intradetrusor injections of botulinum neurotoxin type A (BoNTA) is well documented. Although patient-related outcome measures are highly recommended for monitoring overactive bladder symptoms, no study before has dealt with the question of patient-reported complete continence after BoNTA treatment using validated questionnaires.

Kinematic improvement following Botulinum Toxin-A injection in upper-limb spasticity due to stroke

Background Focal spasticity is a significant motor disorder following stroke, and Botulinum Toxin Type-A (BoNT-A) is a useful treatment for this. The authors evaluated kinematic modifications induced by spasticity, and whether or not there is any improvement following injection of BoNT-A. Methods Eight patients with stroke with upper-limb spasticity, showing a flexor pattern, were evaluated using kinematics before and after focal treatment with BoNT-A. A group of sex- and age-matched normal volunteers acted as a control group. Results Repeated-measures ANOVA showed that patients with stroke performed more slowly than the control group. Following treatment with BoNT-A, there was a significant improvement in kinematics in patients with stroke, while in the control group, performance remained unchanged. Conclusions Focal treatment of spasticity with BoNT-A leads to an adaptive change in the upper limb of patients with spastic stroke.

Lethal toxin of Clostridium sordellii is associated with fatal equine atypical myopathy

The lethal toxin of Clostridium sordellii (TcsL) evokes severe, mostly fatal disease patterns like toxic shock syndrome in humans and animals. Since this large clostridial toxin-induced severe muscle damaging when injected intramuscularly into mice, we hypothesized that TcsL is also associated with equine atypical myopathy (EAM), a fatal myodystrophy of hitherto unknown etiology. Transmission electron microscopy revealed skeletal and heart muscles of EAM-affected horses to undergo degeneration ultrastructurally similar to the damage found in TcsL-treated mice. Performing immunohistochemistry, myofibers of EAM-affected horses specifically reacted with sera derived from horses with EAM as well as an antibody specific for the N-terminal part of TcsL, while both antibodies failed to bind to the myofibers of either healthy horses or those with other myopathies. The presence of TcsL in myofibers of horses with EAM suggests that it plays a role as trigger or even as lethal factor in this disease.

Botulinum Toxin Type A for the Treatment of Equinus Deformity in to Patients With Mucopolysaccharidosis Type II

Mucopolysaccharidoses are lysosomal storage disorders that are caused by a deficiency in the enzymes that degrade glycosaminoglycans. The accumulation of glycosaminoglycans affects multiple systems, resulting in coarse facial features, short stature, organomegaly, and variable neurological changes from normal intelligence to severe mental retardation and spasticity. Effects on the musculoskeletal system include dysostosis multiplex, joint stiffness, and muscle shortening. This article reports 2 patients with mucopolysaccharidosis type II (Hunter syndrome) who showed progressive equinus deformity of the feet. Both patients were treated with intramuscular botulinum toxin type A injections in the gastrocnemius and the soleus muscles, followed by serial casting. In both patients, passive range of motion, muscle tone, and gait performance were significantly improved. Botulinum toxin type A injections followed by serial casting are a therapeutic option for contractures in patients with mucopolysaccharidosis. However, the long-term effects and the effect of application in other muscles remain unknown.

Genetic and functional characterization of the NanA sialidase from Clostridium chauvoei

ABSTRACT: Clostridium chauvoei is the causative agent of blackleg, a wide spread serious infection of cattle and sheep with high mortality. In this study we have analyzed the sialidase activity of the NanA protein of C. chauvoei and cloned the sialidase gene nanA. Sialidase is encoded as a precursor protein of 722 amino acids with a 26 amino acid signal peptide. The mature sialidase has a calculated molecular mass of 81 kDa and contains the carbohydrate binding module 32 (CBM32, or F5/8 type C domain), the sialic acid binding module CBM40 and the enzymatically active sialidase domain found in all pro- and eukaryotic sialidases. Sialidase activity does not require the CBM32 domain. The NanA protein is secreted by C. chauvoei as a dimer. The nanA gene was found to be conserved and sialidase activity was found in C. chauvoei strains isolated over a period of 50 years from various geographical locations. Antiserum directed against a recombinant 40 kDa peptide containing CBM40 and part of the enzymatically active domain of NanA neutralized the secreted sialidase activity of all C. chauvoei strains tested.

Rapid cytopathic effects of Clostridium perfringens beta-toxin on porcine endothelial cells

Clostridium perfringens type C isolates cause fatal, segmental necro-hemorrhagic enteritis in animals and humans. Typically, acute intestinal lesions result from extensive mucosal necrosis and hemorrhage in the proximal jejunum. These lesions are frequently accompanied by microvascular thrombosis in affected intestinal segments. In previous studies we demonstrated that there is endothelial localization of C. perfringens type C beta-toxin (CPB) in acute lesions of necrotizing enteritis. This led us to hypothesize that CPB contributes to vascular necrosis by directly damaging endothelial cells. By performing additional immunohistochemical studies using spontaneously diseased piglets, we confirmed that CPB binds to the endothelial lining of vessels showing early signs of thrombosis. To investigate whether CPB can disrupt the endothelium, we exposed primary porcine aortic endothelial cells to C. perfringens type C culture supernatants and recombinant CPB. Both treatments rapidly induced disruption of the actin cytoskeleton, cell border retraction, and cell shrinkage, leading to destruction of the endothelial monolayer in vitro. These effects were followed by cell death. Cytopathic and cytotoxic effects were inhibited by neutralization of CPB. Taken together, our results suggest that CPB-induced disruption of endothelial cells may contribute to the pathogenesis of C. perfringens type C enteritis.

Real-time multiplex PCR assays for reliable detection of Clostridium perfringens toxin genes in animal isolates

Typing of Clostridium perfringens strains by PCR-based determination of toxin genes proved to be a reliable method for diagnosis of enterotoxaemia in various animal species. We report the establishment and validation of three real-time fluorogenic (TaqMan) multiplex PCRs for the detection of C. perfringens alpha-, beta-, beta2-, epsilon-, entero- and iota-toxin genes. The composition of the PCRs was chosen with regard to robustness of the assays and in order to increase sensitivity compared to the conventional simplex PCRs. The combination of probe dyes selected for the real-time assays (FAM/TAMRA, Cy-5/BHQ-2 and VIC/TAMRA) as well as the designation of the chromosome-borne alpha-toxin as internal positive control allowed the creation of highly specific and sensitive, as well as time and cost effective PCRs. One hundred and three strains of C. perfringens isolated in Switzerland derived from clinical or suspected cases of enterotoxaemia in 10 different animal species were tested. The toxin genotypes were in agreement in both the conventional PCRs and the newly designed multiplex PCRs. Furthermore, the real-time PCR carried out as simplex allows to quantitate the copy numbers of plasmid-borne toxin genes in relation to the chromosomally located alpha-toxin gene.

Clean intermittent self-catheterization after botulinum neurotoxin type A injections: short-term effect on quality of life

OBJECTIVE: To investigate the hypothesis that the need for clean intermittent self-catheterization after botulinum neurotoxin type A injections is outweighed by the efficacy of this treatment, so that clean intermittent self-catheterization is not a burden for patients with refractory idiopathic detrusor overactivity. METHODS: Women undergoing intradetrusor injections of 200 units botulinum neurotoxin type A for refractory idiopathic detrusor overactivity were evaluated prospectively. Clean intermittent self-catheterization was discussed with all patients and its possible need after botulinum neurotoxin type A treatment. As indicator of quality of life, lower urinary tract symptom distress and effect on daily activities were assessed using the validated Urogenital Distress Inventory (UDI-6) and Incontinence Impact Questionnaire (IIQ-7) before and 4 weeks after receiving botulinum neurotoxin type A injections. RESULTS: Mean age of the 65 women was 51 years, and all voided spontaneously before botulinum neurotoxin type A injections. After botulinum neurotoxin type A treatment, 28 (43%) required clean intermittent self-catheterization. Mean UDI-6 and IIQ-7 scores reduced from 61 to 33 (P<.001) and 62 to 30 (P<.001) in women performing clean intermittent self-catheterization and from 60 to 28 (P<.001) and 64 to 25 (P<.001) in those who did not, respectively. Comparison of quality of life in women performing clean intermittent self-catheterization and in those who did not revealed no significant differences before and after botulinum neurotoxin type A treatment. CONCLUSION: Clean intermittent self-catheterization after botulinum neurotoxin type A intradetrusor injections did not impair quality of life in appropriately informed and selected women in the short term. All patients should be informed of the potential need for clean intermittent self-catheterization after botulinum neurotoxin type A injections, and a willingness to do so should be a prerequisite for this still unlicensed off-label treatment.

What a patient with refractory idiopathic detrusor overactivity should know about botulinum neurotoxin type a injection

PURPOSE: We documented the effects of intradetrusor injections of botulinum neurotoxin type A (Botox(R)) for refractory idiopathic detrusor overactivity so that prospective patients maybe properly informed about possible improvement in quality of life, the duration of interinjection intervals and the risk of clean intermittent self-catheterization. MATERIALS AND METHODS: A total of 81 consecutive patients with refractory idiopathic detrusor overactivity treated with intradetrusor injections of 200 U botulinum neurotoxin type A at 20 sites per injection course were evaluated in this prospective, nonrandomized, open label cohort study. The primary outcome was changes in quality of life, as assessed by the short form of the Urogenital Distress Inventory and the Incontinence Impact Questionnaire before and after treatment. Secondary outcomes were the interinjection interval and the need for clean intermittent self-catheterization. RESULTS: After intradetrusor botulinum neurotoxin type A injections there was significant improvement in quality of life, which was sustained after repeat injections. Mean Urogenital Distress Inventory and Incontinence Impact Questionnaire scores decreased from 56 to 26 and 59 to 21 after injection 1 in 81 patients, from 52 to 30 and 51 to 24 after injection 2 in 24, from 40 to 19 and 43 to 17 after injection 3 in 13, from 44 to 17 and 61 to 15 after injection 4 in 6 and from 51 to 17 and 63 to 14 after injection 5 in 4, respectively. The median interinjection interval was 15, 12, 14 and 13 months between injections 1 and 2, 2 and 3, 3 and 4, and 4 and 5, respectively. Considering a post-void residual urine of greater than 100 ml with lower urinary tract symptoms as the indication for clean intermittent self-catheterization, the overall clean intermittent self-catheterization rate after treatment was 43%. CONCLUSIONS: Intradetrusor botulinum neurotoxin type A injections for refractory idiopathic detrusor overactivity significantly improved quality of life. This effect was sustained after repeat injection. More than 2 of 5 patients with refractory idiopathic detrusor overactivity required clean intermittent self-catheterization after botulinum neurotoxin type A injections and all prospective patients should be informed about this.

Suburothelial myofibroblasts in the human overactive bladder and the effect of botulinum neurotoxin type A treatment

BACKGROUND: An increasing body of evidence suggests a possible role of suburothelial myofibroblasts (MFs) in bladder mechanosensation and in the pathophysiology of detrusor overactivity (DO). OBJECTIVE: To determine whether markers of MFs, including gap junction protein connexin43 (Cx43) and c-kit have altered immunohistochemical expression in the suburothelium of patients with neurogenic DO (NDO) or idiopathic DO (IDO) and whether this is affected by successful treatment of DO with botulinum neurotoxin type A (BoNTA). DESIGN, SETTING, AND PARTICIPANTS: Patients with NDO (n=10) or IDO (n=11) were treated in a single-centre, open-label study of intradetrusor BoNTA injections. Control tissue was obtained from 10 patients undergoing pelvic-floor repair procedures who had no overactive bladder (OAB) symptoms. This study is registered with ClinicalTrials.gov, number NCT00662064. INTERVENTIONS: Bladder biopsies performed with flexible cystoscopes were obtained from control subjects and from NDO and IDO patients before BoNTA treatment and at 4 wk and 16 wk after treatment. They were studied with quantitative immunofluorescence using antibodies to connexin 43 (Cx43), vimentin, and c-kit. MEASUREMENTS: Differences in Cx43, vimentin, and c-kit immunoreactivity between control subjects and NDO or IDO patients (primary outcomes). Changes in NDO or IDO, Cx43 immunoreactivity, and c-kit immunoreactivity after BoNTA treatment (secondary outcomes). RESULTS AND LIMITATIONS: Cx43 immunoreactivity was increased in both IDO and NDO patients compared to controls, but remained unchanged after BoNTA treatment. C-kit immunoreactivity was similar in NDO/IDO patients and controls and remained unchanged after BoNTA treatment. CONCLUSIONS: Increased gap junction formation in the suburothelium has been demonstrated in biopsies from humans with DO. It is hypothesised that this change could have a significant role in the pathogenesis of the detrusor abnormality. Successful treatment of NDO or IDO does not appear to be associated with changes in the expression of Cx43 or c-kit on suburothelial MFs.