Epidemiological aspects and diagnosis of botulism in waterfowl

ABSTRACT: Botulism is generally a fatal disease caused by ingestion of neurotoxins produced by Clostridium botulinum. The present study describes the epidemiological, clinical, and laboratory aspects of a type C botulism outbreak in free-living aquatic birds residing in an urban park in Quirinópolis, Goiás, Brazil. Among a population of approximately 80 waterfowl, a total of 30 birds, including ducks (Cairina moschata), teals (Anas platyrhynchos), and geese (Anser cygnoides), died within 10 days. Of these, six birds showed signs of flaccid paralysis of the pelvic limbs, eyelids, neck, and wings. To confirm the suspicion of botulism, four lake water samples, two samples of the feed consumed by the birds, and samples of serum, intestinal content, stomach content, and liver tissue from two teals that died after presenting clinical signs were analyzed. Using bioassay and neutralization with homologous antitoxin in mice, it was possible to detect the presence of botulinum toxin type C in a water sample and in the intestinal content of one of the necropsied teals. Additionally, the presence of C. botulinum type C was identified in the lake water using polymerase chain reaction. Based on the clinical signs and laboratory results, a diagnosis of botulism caused by botulinum toxin type C was confirmed with probable transmission by lake water.

Botulism is a rare paralytic illness resulting from a potent neurotoxin produced by Clostridium botulinum. Botulism in Canada is predominately due to C botulinum type E and affects mainly the First Nations and Inuit populations. The most recent outbreak of botulism in Ontario was in Ottawa in 1991 and was caused by C botulinum type A. We report an outbreak of foodborne type B botulism in Ontario, which implicated home-canned tomatoes. The outbreak was characterized by mild symptoms in two cases and moderately severe illness in one case. The investigation shows the importance of considering the diagnosis of botulism in patients presenting with cranial nerve and autonomic dysfunction, especially when combined with gastrointestinal complaints; it also highlights the importance of proper home canning technique.

Anti-botulism single-shot vaccine using chitosan for protein encapsulation by simple coacervation

Differential effects of sporulation temperature on the high pressure resistance of Clostridium botulinum type E spores and the interconnection with sporulation medium cation contents

Clostridium botulinum, a Gram-positive, anaerobic sporeforming bacteria, is distinguished by its significant clinical applications as well as its potential to be used as bioterror agent. Growing cells secrete botulinum neurotoxin (BoNT), the most poisonous of all known poisons. While BoNT is the causative agent of deadly neuroparalytic botulism, it also serves as a remarkably effective treatment for involuntary muscle disorders such as blepharospasm, strabismus, hemifacial spasm, certain types of spasticity in children, and other ailments. BoNT is also used in cosmetology for the treatment of glabellar lines, and is well-known as the active component of the anti-aging medications Botox® and Dysport®. In addition, recent reports show that botulinum neurotoxin can be used as a tool for pharmaceutical drug delivery. However, BoNT remains the deadliest of all toxins, and is viewed by biodefense researchers as a possible agent of bioterrorism (BT). Among seven serotypes, C. botulinum type A is responsible for the highest mortality rate in botulism, and thus has the greatest potential to act as biological weapon. Genome sequencing of C. botulinum type A Hall strain (ATCC 3502) is now complete, and has shown the genome size to be 3.89 Mb with a G+C content of approximately 28.2%. The bacterium harbors a 16.3 kb plasmid with a 26.8% G+C content—slightly lower than that of the chromosome. Most of the virulence factors in C. botulinum are chromosomally encoded; bioinformatic analysis of the genome sequence has shown that the plasmid does not harbor toxin genes or genes for related virulence factors. Interestingly, the plasmid does harbor genes essential to replication, including dnaE, which encodes the alpha subunit of DNA polymerase III which has close similarity with its counterpart in C. perfringens strain 13. The plasmid also contains similar genes to those that encode the ABC-type multidrug transport ATPase, and permease. The presence of ABC-type multidrug transport ATPase, and permease suggests putative involvement of efflux pumps in bacteriocin production, modification, and export in C. botulinum. The C. botulinum plasmid additionally harbors genes for LambdaBa04 prophage and site-specific recombinase that are similar to those found in the Ames strain of Bacillus anthracis; these genes and their products may play a role in genomic rearrangement. Completion of genome sequencing for C. botulinum will provide an opportunity to design genomic and proteomic-based systems for detecting different serotypes of C. botulinum strains in the environment. The completed sequence may also facilitate identification of potential virulence factors and drug targets, as well as help characterize neurotoxin-complexing proteins, their polycistronic expression, and phylogenetic relationships between different serotypes.

In this study the influence of bovine serum protease inhibitors, trypsin and proteases produced by different types of Clostridium botulinum has been investigated. Trypsin and botulinum proteases had the capability of increasing the toxicity in growing cultures in Clostridium botulinum types A, B and E. Trypsin increased the toxin level to a greater extent than proteases from Clostridium botulinum types A, B, C and F. Protease inhibitors did not influence the toxin formation to any extent compared with the controls. The combined effects of proteases and protease inhibitors on the development of toxin in Clostridium botulinum type B were also investigated by adding proteases and protease inhibitors to the same culture at different time intervals. Protease inhibitors did not reduce the toxicity of the cultures as compared to the controls. Altogether a complex relationship seems to exist between protoxin, toxin, proteases and inhibitors in the culture, and the order and time sequence of addition seem to be of importance. The results obtained in this investigation indicate that proteases of Clostridium botulinum play a part in the formation and/or activation of toxin in growing cultures of proteolytic strains such as Clostridium botulinum types A and B. As to the activation of protoxin and progenitor toxin produced by non-proteolytic Clostridium botulinum types B and E, botulinum proteases showed a marked capability of increasing the toxicity in these cultures. Trypsinization may be valuable for the detection of Clostridium botulinum types A and B in foods, as well as for type E, where it is commonly used.

In this study, a total of 300 honey samples were collected from different sale points in Diyarbakir, Mardin, Batman, Mus, Siirt, Bitlis, and Bingol cities in the Eastern and Southeastern Anatolian regions of Türkiye. The honey samples were analyzed for the presence of Clostridium spp., and Clostridium botulinum types A, B, E, and F. Supernatant filtration and dilution centrifuga- tion methods were used for the detection of Clostridium botulinum spores from honeys. Clostridium botulinum types A, B, E, and F were determined by multiplex real-time polymerase chain reaction. Out of 300 analyzed honey samples, 59 (19.6%) were contaminated with Clostridium spp. It was found that the highest amount of Clostridium spp. contamination belonged to the honey collected from Bingol with 28.5% and the Bitlis city had at lowest 12.5% (p > .05). However, Clostridium botulinum types A, B, E, and F were not detected in any of the samples including those confirmed as Clostridium spp. isolates by polymerase chain reaction. As a result, Clostridium spp. spo- res were determined to be at relatively low levels as none of the samples contained Clostridium botulinum spores. This situation can be considered as a result of the ongoing good production practices in the beekeeping industry in Türkiye. Cite this article as: Guran, H. S., Vural, A., & Erkan, M. E. (2023). Presence of Clostridium spp. and Clostridium botulinum types A, B, E, and F in honey. Acta Veterinaria Eurasia, 49(2), 99-104.

By the use of the electrophoretic casein precipitating inhibition test (CPI-test) the serological relationship between proteolytic enzymes produced by different species within the genera Clostridium and Bacillus has been tested. The proteases produced by Clostridium botulinum types A, B, C, D and F cross-reacted with each other. Clostridium botulinum strain 84 was inhibited by antiproteases produced against Clostridium sporogenes, Clostridium botulinum types C and F (protease F I and F II), but not by antiproteases against Clostridium botulinum types B and F (protease II), Clostridium bifermentans and Clostridium perfringens. The protease of the newly described Clostridium botulinum strain 89 (type G) was inhibited by Clostridium sporogenes antiprotease, but not by any of the other antiproteases. It is not possible to differentiate between Clostridium botulinum, Clostridium sporogenes and Clostridium perfringens by use of serological differentiation of their proteolytic enzymes. The protease of Clostridium bifermentans is not serologically related to any of the species tested in this investigation. Proteases produced by different Bacilli were not inhibited by antiproteases from Clostridium botulinum types B, C and F, Clostridium sporogenes, Clostridium bifermentans, and the two strains of Clostridium perfringens tested. This investigation indicates a serological relationship between proteases from different Clostridium species, but not a serological relationship between proteases produced by the Clostridium species and Bacillus species tested.

Twenty-two lactating Holstein cattle in Tennessee had clinical signs of intoxication with preformed Clostridium botulinum toxin. These signs included weakness, paralysis of the tongue and chest muscles, abdominal breathing, and, in 11 of the 22 cows, death. Differential diagnoses included hypocalcemia, hypomagnesemia, carbohydrate overload, and several toxicoses including mycotoxin, lead, nitrate, organophosphate, atropine or atropine-like alkaloid, and botulism. A diagnosis of botulism by the ingestion of preformed C. botulinum type B toxin was made by eliminating these other diseases, by finding C. botulinum type B spores in 3 bales of round bale barley haylage fed to these cattle, and by isolating preformed type B toxin from 1 of the 3 bales. Confirmation of the toxin type was made by demonstrating mouse lethality by intraperitoneal injection of specimen extracts with neutralization by C. botulinum type B antitoxin. The haylage, harvested green and encased in black plastic bags to facilitate fermentation, was presumably contaminated by the botulinum toxin when fermentation failed to produce enough acid to lower the pH to 4.5, the pH below which C. botulinum growth is inhibited. Farmers and ranchers who use round hay balers to produce haylage should be alert to this potential problem.

Botulism is a rare but life-threatening condition that primarily results from ingestion of food contaminated with the exotoxins produced by Clostridium botulinum. Although uncommon in clinical settings, it is characterized by acute onset, severe manifestations, and a high mortality rate. Outbreaks linked to egg consumption are rarely reported, and cases occurring during pregnancy are even less common, posing unique diagnostic and therapeutic challenges. We report a family cluster of botulism in China that was associated with the consumption of homemade fermented eggs. All affected individuals exhibited symptoms indicative of botulism, including vomiting, dysphagia, restricted eye movement, progressive limb muscle weakness, and other neurological deficits. Electromyography revealed evidence of neuromuscular dysfunction, and laboratory testing confirmed the presence of C. botulinum type A toxin, establishing the diagnosis of foodborne botulism. Both patients received timely supportive care, with one case requiring management during pregnancy. Antitoxin therapy was not administered due to delayed recognition, but both patients recovered fully and were discharged without complications. The pregnant woman subsequently delivered without adverse maternal or neonatal outcomes, and no recurrence was observed during follow-up. Accurate diagnosis of botulism can be challenging and careful epidemiological assessment combined with laboratory confirmation is essential to properly identify and define these cases. These cases underscore the importance of early recognition, timely diagnosis, and prompt treatment in improving patient outcomes, particularly in pregnancy-associated botulism.

Seven distinct strains of Clostridium botulinum (type A to G) each produce a stable complex of botulinum neurotoxin (BoNT) along with neurotoxin-associated proteins (NAPs). Type A botulinum neurotoxin (BoNT/A) is produced with a group of NAPs and is commercially available for the treatment of numerous neuromuscular disorders and cosmetic purposes. Previous studies have indicated that BoNT/A complex composition is specific to the strain, the method of growth and the method of purification; consequently, any variation in composition of NAPs could have significant implications to the effectiveness of BoNT based therapeutics. In this study, a standard analytical technique using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and densitometry analysis was developed to accurately analyze BoNT/A complex from C. botulinum type A Hall strain. Using 3 batches of BoNT/A complex the molar ratio was determined as neurotoxin binding protein (NBP, 124 kDa), heavy chain (HC, 90 kDa), light chain (LC, 53 kDa), NAP-53 (50 kDa), NAP-33 (36 kDa), NAP-22 (24 kDa), NAP-17 (17 kDa) 1:1:1:2:3:2:2. With Bradford, Lowry, bicinchoninic acid (BCA) and spectroscopic protein estimation methods, the extinction coefficient of BoNT/A complex was determined as 1.54 ± 0.26 (mg/mL)(-1)cm(-1). These findings of a reproducible BoNT/A complex composition will aid in understanding the molecular structure and function of BoNT/A and NAPs.

Nucleotide sequence of the gene coding for Clostridium botulinum ( Clostridium argentinense) type G neurotoxin: Genealogical comparison with other clostridial neurotoxins

C1 neurotoxin of Clostridium botulinum strains C-Stockholm (C-ST), C beta-Yoichi, C-468, CD6F, and C-CB19 and type D toxin of strains D-1873 and D-CB16 were purified by gel filtration, ion exchange, and affinity chromatographies. The purified toxins had di-chain structure made of heavy and light chains. The toxins of C beta-Yoichi, C-468, CD6F, and C-CB19 reacted with anti-C-ST heavy chain and anti-C-ST light chain in immunodiffusion tests and enzyme-linked immunosorbent assay, whereas D-CB16 toxin reacted with anti-D-1873 heavy chain and anti-D-1873 light chain. However, C-6813 toxin reacted with anti-D-1873 heavy chain and anti-C-ST light chain but not with anti-C-ST heavy chain or anti-D-1873 light chain immunoglobulin G. These results indicate common antigens in the heavy chains of C-6813 and D-1873 toxins and in the light chains of C-6813 and C-ST toxins. Further, they provide evidence for heterogeneity within type C1 toxin subunits.

Bacteria with inhibitory activity against Clostridium botulinum type C were isolated from 32% of sediment samples (n = 1600) collected from 10 marshes in a northern California wetland over a 12 mo period. Aerobic and anaerobic bacteria with inhibitory activity were isolated from 12% and 23% of the samples, respectively. Bacteria with inhibitory activity were isolated from all 10 study sites and throughout the year. This study demonstrates that bacteria with inhibitory activity against C. botulinum type C occur naturally in wetland sediments.

Use of Preservatives to Delay Toxin Formation by Clostridium botulinum (Type B, Strain Okra) in Vacuum-Packed, Cooked Potatoes