Neurotransmitter vesicle release from human model neurons (NT2) is sensitive to botulinum toxin A.

Botulinum neurotoxins (BoNTs) internalize into nerve terminals and block the release of neurotransmitters into the synapse. BoNTs are widely used as a therapeutic agent for treatment of movement disorders and recently gained more attention as a biological weapon. Consequently, there is strong interest to develop a cell-based assay platform to screen the toxicity and bioactivity of the BoNTs. In this study, we present an in vitro screening assay for BoNT/A based on differentiated human embryonal carcinoma stem (NT2) cells. The human NT2 cells fully differentiated into mature neurons that display immunoreactivity to cytoskeletal markers (ßIII-tubulin and MAP2) and presynaptic proteins (synapsin and synaptotagmin I). We showed that the human NT2 cells undergo a process of exo-endocytotic synaptic vesicle recycling upon depolarization with high K(+) buffer. By employing an antibody directed against light chain of BoNT/A, we detected internalized toxin as a punctate staining along the neurites of the NT2 neurons. Using well-established methods of synaptic vesicle exocytosis assay (luminal synaptotagmin I and FM1-43 imaging) we show that pre-incubation with BoNT/A resulted in a blockade of vesicle release from human NT2 neurons in a dose-dependent manner. Moreover, this blocking effect of BoNT/A was abolished by pre-adsorbing the toxin with neutralizing antibody. In a proof of principle, we demonstrate that our cell culture assay for vesicle release is sensitive to BoNT/A and the activity of BoNT/A can be blocked by specific neutralizing antibodies. Overall our data suggest that human NT2 neurons are suitable for large scale screening of botulinum bioactivity.

Challenging the problem of clostridial identification with matrix-assisted laser desorption and ionization-time-of-flight mass spectrometry (MALDI-TOF MS).

Diverse techniques were applied to effect the identification and classification of isolated clostridial strains. Nevertheless, the correct identification of clostridial strains remains a laborious, time-consuming task which entails a not inconsiderable degree of expertise. In addition to this, traditional methods based on the metabolic properties of the bacteria require rigorously standardized media and growth conditions to assure the attainment of reproducible results. Although DNA-based methods, like the PCR of a species specific gene, are known to yield precise and reproducible results, their degree of effectivity is circumscribed by the fact that even the incidence of a toxin encoding gene is not necessarily linked to nor consequently indicative of the presence of an infectious disease. Moreover, most of these methods postulate an initial assumption concerning the expected bacterial species involved before the choice of PCR primer for use can be made. Consequently, the scope of these methods is restricted to that of targeted analyses. The 16S rDNA sequencing which is assumed to be the gold standard for bacterial classification having the unequivocal advantage of being capable of determining even uncultivable bacteria is nonetheless a time-consuming and costly technique. In the present study we describe the utilization of matrix-assisted laser desorption and ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for whole cell fingerprinting in combination with a dedicated bioinformatic software tool to distinguish between various clostridial species. Total 64 clostridial strains of 31 different species each displayed a mass spectrum unique to the strain involved, to the effect that it was also possible not only to differentiate between the strains examined, but also to establish to which species the individual strains belonged to. Starting with a single colony it was possible to correctly identify a Clostridium species within minutes. It was even possible to identify species which are normally difficult to differentiate by traditional methods, such as C. chauvoei and C. septicum. With the results obtained we were able to assemble a dendrogram of the Clostridium species which showed considerable similarities to dendrograms based upon 16S rDNA sequencing data. To conclude, our findings indicate that, inasmuch as the MALDI-TOF MS technology employed is based on a high-quality reference database, it may serve as an effective tool for the swift and reliable identification and classification of Clostridia.

Evaluation of lateral flow assays for the detection of botulinum neurotoxin type A and their application in laboratory diagnosis of botulism.

Four lateral flow assays (LFAs) were evaluated for the detection of purified botulinum neurotoxin A, toxin complex, and unpurified culture supernatant. They included the BioThreat (Tetracore, Rockville, MD), SMART (New Horizons Diagnostics, Columbia, MD), BADD (ADVNT Biotechnologies, Phoenix, AZ), and RAMP (Response Biomedical, Burnaby, BC, Canada) assays. BioThreat and SMART did not detect the purified toxin. The best sensitivity was achieved with the RAMP test (50 ng mL(-1)). BioThreat and SMART measured as low as 10 ng mL(-1) of the toxin complex. Specificity data differed among the tests. BADD gave false-positive signals with uninoculated bacterial culture medium. BioThreat and RAMP were further evaluated with clinical sample matrices (serum, gastric, and rectum contents from pigs). Because of matrix effects and a generally low positive response, the assays are unsuitable for the direct detection of the toxin. However, the LFAs can be a helpful tool in screening bacterial cultures for toxigenic Clostridium botulinum, if further validated according to the laboratory needs.

Immunomagnetic beads assay for the detection of botulinum neurotoxin types C and D.

An immunomagnetic beads assay for the simultaneous quantification of botulinum neurotoxin types C and D was developed. Specific monoclonal antibodies against the heavy chain of the toxin and affinity-purified biotinylated polyclonal antibodies (pAbs) were used. The antibodies were preincubated with the sample. The complex being formed was then captured by magnetic beads coated with antimouse IgG. Streptavidin-poly-horseradish peroxidase, a signal amplifier, bound to the biotinylated pAb. A maximum sensitivity of approximately 0.3 minimal lethal doses for mice per milliliter was achieved with culture supernatants of both toxin types.

Persistence and mobility of a Clostridium botulinum spore population introduced to soil with spiked compost.

In a recent study it could be shown that compost samples can contain Clostridium botulinum. It was investigated if C. botulinum introduced with compost into botulinum-free soil can persist and be translocated within the soil. Compost was spiked with two C. botulinum type D spore concentrations (10(3) and 10(5) spores g(-1)) and the composts were spread on an experimental site. Over a period of 939 days, samples were taken from the upper (0-5 cm) and the lower (10-30 cm) soil horizons. Physical and chemical as well as microbiological variables were measured. Clostridium botulinum spores were quantified in a culture MPN-PCR assay. On day 757 the last positive sample was obtained in the plots with the lower spore concentration (10(3) g(-1)). The bacteria were never detected in the samples taken from the lower horizons of these plots. Clostridium botulinum persisted over the whole investigation period in the plots which were treated with compost spiked with 10(5) spores g(-1). The concentrations found were between 20 and 20,000 spores g(-1) soil. The bacteria were vertically translocated and could be found in the lower soil horizons (20-2000 spores g(-1) soil) starting 70 days after the compost was spread.

Systemic colonization of clover (Trifolium repens) by Clostridium botulinum strain 2301.

In recent years, cases of botulism in cattle and other farm animals and also in farmers increased dramatically. It was proposed, that these cases could be affiliated with the spreading of compost or other organic manures contaminated with Clostridium botulinum spores on farm land. Thus, soils and fodder plants and finally farm animals could be contaminated. Therefore, the colonization behavior and interaction of the botulinum neurotoxin (BoNT D) producing C. botulinum strain 2301 and the non-toxin producing Clostridium sporogenes strain 1739 were investigated on clover (Trifolium repens) in a field experiment as well as in phytochamber experiments applying axenic and additionally soil based systems under controlled conditions. Plants were harvested and divided into root and shoot parts for further DNA isolation and polymerase chain reaction (PCR) assays; subsamples were fixed for fluorescence in situ hybridization analysis in combination with confocal laser scanning microscopy. In addition, we observed significant differences in the growth behavior of clover plants when inoculated with clostridial spores, indicating a plant growth promoting effect. Inoculated plants showed an increased growth index (shoot size, wet and dry weight) and an enlarged root system induced by the systemic colonization of clover by C. botulinum strain 2301. To target C. botulinum and C. sporogenes, 16S rDNA directed primers were used and to specifically detect C. botulinum, BoNT D toxin genes targeted primers, using a multiplex PCR approach, were applied. Our results demonstrate an effective colonization of roots and shoots of clover by C. botulinum strain 2301 and C. sporogenes strain 1739. Detailed analysis of colonization behavior showed that C. botulinum can occur as individual cells, in cell clusters and in microcolonies within the rhizosphere, lateral roots and within the roots tissue of clover.

Sensitive detection of botulinum neurotoxin types C and D with an immunoaffinity chromatographic column test.

A sensitive and specific immunoassay for the simultaneous detection of Clostridium botulinum type C (BoNT/C) and type D neurotoxin was developed. Goat anti-mouse immunoglobulin G was bound to polyethylene disks in a small disposable column used for this assay. The sample was preincubated together with monoclonal antibodies specific for the heavy chain of BoNT/C and D and affinity-purified, biotinylated polyclonal antibodies against these neurotoxins. This complex was captured on the assay disk. Streptavidin-poly-horseradish peroxidase was used as a conjugate, and a precipitating substrate allowed the direct semiquantitative readout of the assay, if necessary. For a more accurate quantitative detection, the substrate can be eluted and measured in a photometer. Depending on the preincubation time, a sensitivity of 1 mouse lethal dose ml(-1) was achieved in culture supernatants.

[Diagnosis of botulism since 1995. Report of test results]. / Botulismusdiagnose seit 1995. Ein Erfahrungsbericht.

Since 1970 our laboratory is specialized in diagnosis of clostridial diseases, including Clostridium botulinum and botulism. Since 1995, samples from more than 900 suspected botulinal cases were received, mainly in cattle, horses and men. 524 outbreaks were diagnosed as clearly positive by toxin neutralisation; 83 cases remained inconclusive with the toxin neutralisation. The geographical distribution of the positive cases in Germany is demonstrated for cattle and horses. Dispatch and treatment of specimens and interpretation of results are discussed.

[Development of an in vitro assay for the detection of Botulinum Neurotoxins]

Botulism is a severe, often lethal intoxication, which affects man and animal. It is caused by the neurotoxins (BoNT) of Clostridium botulinum, an anaerobic spore-former. At present the toxin types A-G have been identified. The extreme toxicity of these substances still make the animal testing inevitable. No alternative method could reach the sensitivity of the mouse bioassay. Based on the investigations presented, a serological test shall be developed, which concentrates the sample and detects the toxin in one step. Preliminary results of an in vitro test detecting BoNT/D are discussed.