Search Results (164)

Botulism is a neuroparalytic disease caused by exposure to botulinum neurotoxins produced by anaerobic spore-forming bacteria of the genus Clostridium. This disease occurs in both humans and wild and domestic animals, and is currently becoming an increasingly serious problem worldwide due to high animal mortality and economic losses. The clinical signs observed during the progression of botulism are nonspecific and difficult to unequivocally associate with this disease entity. The aim of this study is to present laboratory diagnostics of suspected botulism cases reported in Poland in 2022–2024, as well as to present the challenges encountered during laboratory investigations. The material for the study consisted of samples of liver, serum, digestive tract, feed, feces, straw, and water from drinking lines, sent to the National Veterinary Research Institute (NVRI) in relation to thirteen suspected cases of botulism, predominantly reported in poultry, but also in mink and cattle farms. The samples were analyzed using a mouse bioassay and conventional culture methods, as well as real-time PCR methods aimed at detecting the ntnh and bont genes, which determine the production of botulinum neurotoxins. Of the thirteen suspected cases analyzed, ten were confirmed by the detection of botulinum toxin (BoNTs) and/or the presence of the ntnh and bont genes in the tested material. Based on the results obtained, it was concluded that botulinum toxin type C was the etiological factor of botulism poisoning in most of the analyzed cases. In one case reported in cattle, poisoning occurred as a result of the mosaic variant of BoNT D/C. Due to the nonspecific signs of botulism and the time required for them to appear, laboratory diagnostics play a key role in detecting the disease. However, this process is complicated due to the high heterogeneity observed among Clostridium spp. strains, as well as difficulties encountered during the isolation of the microorganism and the possibility of loss of toxin-producing capacity at later stages of analysis. Full article

Botulinum neurotoxins (BoNTs), among the most potent biological toxins, rely on co-produced nontoxic proteins to survive harsh gastrointestinal conditions and achieve efficient systemic dissemination after oral exposure. Recent structural and functional studies have revealed how BoNTs bind to the nontoxic non-hemagglutinin (NTNH) factors to engage in interactions with either OrfXs/P47 or hemagglutinins (HAs) components for systemic dissemination. This review synthesizes recent findings that elucidate the molecular basis of NTNH-specific anchoring to the HA70 triskelion-like element or to the host protease-activated form of OrfX2, thereby highlighting divergent pathways that enhance oral toxicity. We also discuss current perspectives on the molecular mechanisms through which BoNTs, in cooperation with associated nontoxic proteins, are absorbed from the intestine. Full article

Raw meat-based diets (RMBDs) have become increasingly popular among pet owners, despite well-documented risks of contamination with pathogenic bacteria capable of causing severe illness in companion animals. This report describes a fatal case of botulism in a 3-year-old female Labrador Retriever weighing 37 kg that was fed exclusively RMBD. The dog presented with acute-onset flaccid paralysis of the limbs approximately 48 h after possible ingestion of decomposing raw meat discarded in household waste. Supportive therapy, including fluid administration, nutritional support and eventual mechanical ventilation was provided. However, the patient developed progressive respiratory failure and died. The presence of Clostridium botulinum type C neurotoxin was confirmed in the dog serum by neutralization test in mice. The case suggests RMBD as a potential source of botulism toxins, particularly when derived from improperly stored meat products. The findings underscore the importance of detailed dietary history in dogs presenting with acute flaccid paralysis and reinforce the need for heightened awareness regarding the microbiological risks associated with raw feeding practices. Full article

Plasticity is fundamental to the development, strengthening, and maintenance of healthy synaptic connections and recovery from injury in both the central and peripheral nervous systems. Yet, the processes involved are poorly understood. Herein, using a combination of patch-clamp electrophysiology and immuno-fluorescence confocal microscopy in adult mice, it is shown that blockade of synaptic transmission at submandibular ganglion junctions exposed to botulinum neurotoxin type A was accompanied by a rapid and striking decline in the abundance of synaptic vesicle markers—SV2, vesicle-associated membrane protein 2, and vesicular acetylcholine transporter—plus SNAP-25 (cleaved and intact) and postsynaptic α7 nicotinic acetylcholine receptors. Such alterations by the neurotoxin of parasympathetic synapses contrast starkly with the stability of postsynaptic proteins at nearby skeletal neuromuscular junctions. Both neurotransmission and the expression of SV2 and α7 nicotinic acetylcholine receptors remained depressed for 4 weeks, with full recovery of synaptic function delayed for more than 8 weeks. These novel findings may explain the relatively slow recovery of autonomic function after botulism or following therapeutic injections to alleviate hypersecretory disorders. Full article

Clostridial neurotoxins, botulinum neurotoxins (BoNTs), and tetanus neurotoxin (TeNT) are potent toxins responsible for severe diseases, botulism and tetanus, respectively. BoNTs associate with non-toxic proteins (non-toxic non-hemagglutinin, hemagglutinins, and OrfXs), which protect BoNTs against acidic pH and protease degradation and facilitate BoNT passage through the intestinal barrier. TeNT enters motor neurons and undergoes a retrograde axonal transport until the target inhibitory interneurons in the central nervous system. BoNTs and TeNT recognize specific cell surface receptors which consist of complex sets of protein(s)-glycan-gangliosides and determine specific cell entry pathways. Recent data on structural and functional investigations of BoNT and TeNT receptors bring a better understanding of toxin trafficking in the host and entry into target neuronal cells, which is useful for the development of updated strategies of prevention and treatment of the corresponding diseases. Since clostridial neurotoxins, notably BoNTs, are important therapeutic tools, detailed knowledge of their activity opens the way of the development of engineered molecules for specific clinical applications. Full article

Background: Botulism is a rare but potentially fatal neuroparalytic illness caused by Clostridium botulinum neurotoxins (BoNTs). While adult cases usually result from foodborne exposure or wound infection, intestinal colonization is exceedingly uncommon. Diagnosis can be delayed by overlap with other neuromuscular syndromes, and confirmation requires specialized assays. Anti-GQ1b antibodies, classically associated with Miller–Fisher syndrome (MFS), have rarely been reported in confirmed botulism, raising questions about shared pathophysiology. Case Presentation: We describe an adult patient with acute dyspnea, xerostomia, and cranial neuropathies. No foodborne source was identified, but intestinal colonization of BoNT/B toxigenic Clostridium botulinum was confirmed by stool enrichment and mouse lethality bioassay. The patient improved promptly following heptavalent antitoxin. Unexpectedly, anti-GQ1b antibodies were detected during recovery, a finding typically linked to MFS rather than botulism. Discussion: This case illustrates the diagnostic challenges of sporadic cases of botulism, especially when respiratory compromise and autonomic dysfunction dominate the initial presentation. The autoantibodies finding raises the possibility of molecular mimicry, whereby toxin–ganglioside interactions expose neuronal epitopes and trigger an immune response. While causality cannot be proven, the overlap between botulism and GQ1b-positive neuropathies merits further investigation. Conclusions: Clinicians should maintain high suspicion for botulism in adults with acute dyspnea, especially when associated with cranial neuropathies, even in the absence of foodborne exposure. Anti-ganglioside antibodies in this context should be interpreted with caution, as they do not exclude botulism but may highlight immunological overlap with autoimmune neuropathies. Full article

Botulinum neurotoxin serotype A (BoNT/A) is the most potent known neurotoxin. While its light chain (LC) catalytic domain is a prime target for next-generation vaccines and therapeutics, the functional differences among BoNT/A subtype LCs (A1, A2, A3) remain to be definitively characterized, despite notable sequence variation. This work aimed to systematically compare the proteolytic activity and immunoprotective efficacy of recombinant BoNT/A1-LC, A2-LC, and A3-LC. Recombinant A1-LC-His, A2-LC-His, A3-LC-His, and A3-LC-Twin-Strep proteins were expressed in Escherichia coli (E. coli) and purified with affinity chromatography. Their proteolytic activity was assessed via in vitro SNAP-25 cleavage assays. The protective potency of these antigens was evaluated in a mouse model. In vitro cleavage assays revealed a substrate cleavage efficiency order of A2-LC > A1-LC > A3-LC. In vivo, both A1-LC and A2-LC immunization conferred robust, broad protection against high-dose challenges with all three toxin subtypes. In stark contrast, A3-LC provided only minimal protection against its homologous toxin and none against heterologous subtypes. Crucially, the functional deficit of A3-LC was confirmed to be an intrinsic property, as the A3-LC-TS variant, designed to exclude tag-specific interference, exhibited comparable low efficacy. According to structural research, A3-LC’s compromised function may be caused by a four-amino-acid loss. The inferior performance of A3-LC is inherent to its primary structure. This work identified A1-LC or A2-LC as the potential proteolytic activity molecule and vaccine antigen by demonstrating functional differences among BoNT/A subtype LCs. These findings provide crucial insights for developing subtype-specific countermeasures against botulism. Full article

Foodborne botulism is a potentially fatal disease caused by Clostridium botulinum neurotoxins (BoNTs). These spore-forming bacteria are ubiquitous in the environment and can contaminate various food products, especially raw vegetables. During the preparation of home-made preserves, favorable conditions of anaerobiosis, temperature, salinity, and pH can lead to spore germination and toxin production. BoNTs can reach neuromuscular junctions where they block the release of acetylcholine. In this study, we present a case of foodborne botulism associated with the consumption of chili peppers preserve containing BoNT/B. The isolated strains were characterized through Whole Genome Sequencing, confirming the strains involved in the outbreak. This work increases the understanding of the epidemiology and the ecology of C. botulinum, highlighting the importance of raising medical awareness and making timely clinical diagnoses for the effective management of botulism outbreaks. Full article

Botulinum neurotoxins (BoNTs), produced by the anaerobic spore-forming bacterium Clostridium botulinum, are among the most potent known biological toxins. BoNTs cause lethal botulism via contaminated food, wound infections, or infant intestinal colonization, posing significant threats to public health. Although the mouse bioassay is still being considered as the gold standard for detecting BoNTs, its drawbacks, including the lengthy experimental duration, high costs, and ethical issues, highlight the urgent need to develop alternative methods to fulfill the detection requirements. In recent years, frequent botulism poisoning incidents haves put forward higher requirements for detection technology. On-site detection is expected to be rapid and immediate, while laboratory detection requires high sensitivity and serotype discrimination capabilities. This review comprehensively introduces current detection approaches, including mouse bioassay, cell-based assays, immunological methods, endopeptidase–mass spectrometry, biosensors, chromatography, and mass spectrometry techniques. Notably, cell-based assays have been used for the potency testing of commercialized botulinum toxin type A and are considered the most promising alternative to the mouse bioassay. Biosensors based on nanomaterials demonstrate advantages in real-time detection due to their rapid response and portability, while endopeptidase–mass spectrometry achieves high sensitivity and effective serotype identification by specifically recognizing toxin-cleaved substrates. Future works shall aim to completely replace MBA, developing a detection system suitable for multiple scenarios such as clinical diagnosis, food safety monitoring, and environmental monitoring. The detection methods should also have matrix compatibility and serotype discrimination capabilities. Full article

The neurotoxin BoNT/B2 is the predominant Clostridium parabotulinum subtype in foodborne and infant botulism cases in Spain. This study characterizes a novel case of foodborne botulism in Spain caused by a dual-toxin A1(B5) strain. A 64-year-old male presented with acute, progressive flaccid paralysis including diplopia, dysphagia, and respiratory failure. Although botulism was not initially suspected, the patient recovered with supportive care and without antitoxin administration. Genomic characterization confirmed the presence of both bont/A1 and silent bont/B5 genes. The bont/A1 gene was associated with an orfX+ neurotoxin gene cluster, while the silent bont/B5 gene was in an ha+ cluster. Phylogenetic analysis of both bont/A1 and bont/B5 sequences showed 100% amino acid identity, respectively, to previously reported A1(B5) strains (e.g., CDC_69094, FE9504ACG). Multi-locus sequence typing (MLST) assigned the ST10, a genotype previously undetected in Spanish botulism cases, yet found in other European countries. This case highlights the importance of considering botulism in differential diagnosis due to its varied presentation and the significance of timely laboratory confirmation for effective management. The identification of this dual-toxin BoNT/A1(B5) orfX+/ha+ ST10 strain expands our understanding of C. botulinum epidemiology and genetic diversity in Spain. Full article

Food-borne botulism is a rare but serious disease caused by ingestion of botulinum neurotoxin pre-formed in food by Clostridium botulinum. Between 2006 and 2009, no foodborne botulism cases were reported in the UK. However, the period from 2010 to 2024 saw 13 cases, encompassing seven separate incidents and two outbreaks, with no reported fatalities. Cases were predominantly linked to imported, home-made, and artisanal foods, occasionally to commercial products. Diagnostic and public health challenges include delayed clinical diagnosis, delayed sample collection, inadequate specimen volumes, and the frequent unavailability of suspected food sources, hampering epidemiological investigations. The UK has an extremely low incidence of foodborne botulism with an estimated rate of 0.001 cases per 100,000 people per year, but despite this low occurrence, food botulism remains a public health emergency as it requires timely treatment and rapid reactive intervention to be undertaken by multiple regulatory agencies. Continuous professional training of medical staff, up-to-date clinical guidance, rapid diagnostic, and food investigations are essential for optimising patient outcomes and prevention. Full article

Botulism is a severe muscle paralysis disease mediated by the botulinum toxin. Here, we reported a foodborne botulism case caused by Clostridium botulinum subtype A5(b3) from self-packaged vacuum spicy rabbit heads. Treatment for this case was delayed due to misdiagnosis and insufficient diagnostic capacity in three hospitals, which resulted in progressive clinical deterioration, and eventually, the patient was transferred to Shandong Public Health Clinical Center for specialized therapy. The case was suspected as foodborne botulism by the Qilu Medical-Prevention Innovation Integration pathway and multi-disciplinary consultation. An epidemiological investigation and laboratory confirmation revealed that the botulinum neurotoxin originated from vacuum-packaged spicy rabbit heads distributed via interprovincial cold chain logistics. After treatment with botulism antiserum, the patient’s condition significantly improved, and they were discharged after recovery. We revealed that this foodborne botulism outbreak was caused by the Clostridium botulinum A5(b3) subtype from food by whole-genome sequencing and SNP typing. All the strains belonged to Group I carrying the botulinum neurotoxin gene classified as the ha cluster. Toxin A was confirmed by MBA and other methods, while toxin B was non-functional due to the truncated bont/B gene. Other virulence genes and antibiotic resistance genes were also detected. Our findings indicate that self-packaged vacuum meat products represent an emerging risk factor for botulism transmission when stored improperly. Importantly, the recurrent misdiagnosis in this case underscored the urgent need to enhance the training of healthcare professionals in medical institutions to improve the diagnostic accuracy and clinical management of botulism. Full article

Clostridium botulinum produces one of the most potent biological toxins and causes botulism, a rare but potentially fatal neuroparalytic disease. In 2014, a foodborne botulism case was reported in Korea, and a strain (CB-2014001) was isolated. Initial characterization identified it as a BoNT/B-producing strain based on mouse bioassay and conventional PCR. However, subsequent genomic analysis revealed the presence of dual BoNT gene clusters, bont/B and bont/F, corresponding to subtypes B5 and F2, respectively. Therefore, we aimed to analyze the genetic characteristics and toxin expression profiles of the isolated strain. The strain showed high sequence identity with Bf-type strains such as CDC 3281 and An436. Functional assays confirmed simultaneous expression of both BoNT/B and /F toxins at 35 °C, and temperature-dependent assays revealed predominant expression of BoNT/F at 30 °C and BoNT/B at 37 °C, indicating that toxin expression is influenced by environmental temperature. These findings highlight the potential for differential pathogenicity based on culture conditions and underscore the importance of developing diagnostic tools capable of detecting multiple bont genes. To our knowledge, this is the first report of a dual-toxin-producing C. botulinum strain associated with foodborne botulism in Korea, providing important insights into botulism diagnosis, treatment strategies, and public health preparedness. Full article

Recombinant monoclonal antibody (mAb) botulinum neurotoxin (BoNT) antitoxins, consisting of three mAbs that bind non-overlapping epitopes, are highly potent. However, the three-mAb mixtures pose unique development and manufacturing challenges. Combining even more mAbs to create multivalent antitoxin drugs multiplies those challenges. We previously reported that a single tri-epitopic IgG1-based mAb (TeAb) containing the variable domains of the three parental BoNT/A mAbs and an Fc was as potent as the combination of three IgGs in the mouse neutralization assay (MNA). Here, we extended the tri-epitopic strategy to three other BoNT serotypes. Each TeAb (TeAb-B for BoNT/B, TeAb-E for BoNT/E, and TeAb-F for BoNT/F) binding was measured using fluorescence-activated cell sorting and flow fluorimetry, and the potency was tested in the MNA. The three TeAbs displayed binding affinities that were the same within error of the parental IgGs for each epitope, and all had higher avidity to each serotype of BoNT than that of the parental mAbs. The potency of the BoNT/B, BoNT/E, and BoNT/F TeAbs was similar to the combinations of the three parental IgGs binding BoNT/B, BoNT/E, and BoNT/F in the MNA. We now have four examples of a single TeAb recapitulating the affinity and in vivo potency of a three-mAb antitoxin. The tri-epitopic strategy could be applied to streamline the production and bioanalytics of antibody drugs where three-mAb binding is required for activity. Full article

Botulinum neurotoxins (BoNTs) are the most potent toxins on Earth and are classified as Category A biological agents. BoNTs lead to paralysis in humans and cause botulism. Antibody therapeutics can effectively treat toxin-mediated infectious diseases. In this study, we generated a pharmaceutical humanized monoclonal antibody (HZ45 mAb) to prevent or treat botulism. HZ45 binds to the heavy chain receptor (HCR) domain of the toxin, preventing the toxin from entering the cell. The mAb was produced using hybridoma technology and phage display. We evaluated HZ45 mAb for the neutralization of BoNT serotype A (BoNT/A) in mice and rabbits. The survival results showed that pretreatment with HZ45 mAb provided 100% protection at a dose of 0.1 mg per mouse against a maximum of 100 LD₅₀ of BoNT/A. To assess the therapeutic efficacy of HZ45 mAb in New Zealand white rabbits (NZWs), a 5 mg dose was administered 4 or 8 h after challenge with 10 LD₅₀. The results indicated that 5 mg of HZ45 could treat the NZWs within 8 h after exposure to 10 LD₅₀ botulinum. Consequently, in an in vivo context, including mice and rabbits, HZ45 mAb could protect against botulinum type A intoxication. Full article