Search Results (38)

Background/Objectives. Bovine botulism, although relatively rare, presents significant economic losses due to high mortality rates and restrictions on livestock product trade. Vaccination remains the most effective strategy for preventing botulism-related mortality. This study evaluated the efficacy of a bivalent recombinant vaccine targeting the C-terminal portion of the heavy chain (Hc) of botulinum neurotoxin serotype C (BoNT/C) (Hc BoNT/C) and botulinum neurotoxin serotype D (BoNT/D) (Hc BoNT/D) in inducing neutralizing antibodies against these toxins and their mosaic variants BoNT/CD and BoNT/DC in cattle. This comparison aims to improve the design of an optimal recombinant vaccine for preventing bovine botulism caused by the most common serotypes. Methods. Twenty, four-month-old Holstein Friesian calves were randomly assigned to two groups of ten animals: vaccinated group and control group. Sera were collected at various time points to assess antibody titers using ELISA and neutralizing antibody titers using a mouse protection assay. Neutralizing antibody titers were compared to those obtained with a commercially available toxoid vaccine. Results. The recombinant vaccine elicited significant increases in anti-HcBoNT/C and anti-HcBoNT/D IgG antibody levels in vaccinated animals compared to controls animals with no adverse effects. Specifically, post-vaccination, the calves showed no local reactions (swelling, warmth) or behavioral changes suggestive of systemic illness. Neutralizing antibody titers against BoNT/C and BoNT/D were significantly higher in the recombinant vaccine group compared to the toxoid vaccine group. However, the recombinant vaccine showed lower neutralizing activity against BoNT/DC compared to the toxoid vaccine. Conclusions. The bivalent recombinant vaccine demonstrated promising immunogenicity in cattle, inducing high neutralizing antibody titers against BoNT/C and BoNT/D. While effective against these toxins, the lower efficacy against BoNT/DC highlights the need for further research to optimize the vaccine formulation, potentially by incorporating a BoNT/DC Hc component, to provide broader protection against bovine botulism. Full article

Background: Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, are potent protein toxins that can cause botulism, which leads to death or neuroparalysis in humans by targeting the nervous system. BoNTs comprise three functional domains: a light-chain enzymatic domain (LC), a heavy-chain translocation domain (HC_N), and a heavy-chain receptor-binding domain (HC_C). The HC_C domain is critical for binding to neuronal cell membrane receptors and facilitating BoNT internalization via endocytosis. Accordingly, it may serve as a vaccine candidate, inducing anti-BoNT-neutralizing antibodies in animals. Here, we aimed to develop a vaccine capable of simultaneously defending against both BoNT/A and B. Methods: We combined the HC_C domains of botulinum neurotoxin type A (BoNT/A) and botulinum neurotoxin type B (BoNT/B) in Escherichia coli to produce a recombinant protein (rHC_CB-L-HC_CArHCcB) that offers dual protection against both toxins by inhibiting their receptor binding. To evaluate the efficacy of the vaccine, mice were immunized intramuscularly with rHC_CB-L-HC_CA plus alum thrice at 2-week intervals, followed by the assessment of immunogenicity and protective efficacy. Results: The antibody titer in mice immunized with rHC_CB-L-HC_CA was significantly higher than that in mice immunized with alum alone, protecting them from the lethal challenges of BoNT/A (10⁵ 50% lethal dose, LD₅₀) and B (10³ LD₅₀). Conclusion: These findings suggest that rHC_CB-L-HC_CA may simultaneously be an effective vaccine candidate against BoNT/A and B. Full article

The discovery of microbial toxins as the primary factors responsible for disease manifestations and the discovery that these toxins could be neutralised by antitoxins are linked to the birth of immunology. In the late 19th century, the serum or plasma of animals or patients who had recovered from infectious diseases or who had been immunised with a relevant antigen began to be used to treat or prevent infections. Before the advent of widespread vaccination campaigns, antitoxins played a key role in the treatment and prevention of diseases such as diphtheria and tetanus. A significant reduction in mortality following the introduction of antitoxins confirmed their efficacy. Serum therapy remains an important measure for post-exposure prophylaxis and for the treatment of unvaccinated or incompletely vaccinated patients. For the botulinum toxin, antitoxin therapy continues to be the sole available treatment. The manuscript contains a summary of the most important information on the passive immunoprophylaxis used in the treatment of diphtheria, tetanus, and botulism, all representing diseases in which symptoms are driven by the activity of highly potent bacterial toxins. Full article

The cluster of genes determining the production of botulinum toxins is an attribute of not only the Clostridium botulinum species. This cluster is also found in other members of the Clostridium genus, such as C. baratii, C. butyricum, and C. sporogenes. The occurrence of a botulinum-like cluster has also been recorded in strains of other genera, i.e., Enterococcus faecium, as well as in a Gram-negative species isolated from freshwater sediments; however, the biological activity of bont-related genes has not been noted. It can be said that the mentioned species have a dual nature. Another species with a dual nature is C. butyricum. This bacterium is a common human and animal gut commensal bacterium and is also frequently found in the environment. Although non-toxigenic strains are currently used as probiotics in Asia, other strains have been implicated in pathological conditions, such as botulism in infants or necrotizing enterocolitis in preterm neonates. Additionally, C. baratii strains are rare opportunistic pathogens associated with botulism intoxication. They have been isolated from food and soil and can be carried asymptomatically or cause botulism outbreaks in animals and humans. In addition to the mentioned clostridia, the other microorganisms considered as non-toxigenic have also been suspected of carrying botulinum cluster Gram-negative bacteria, such as Chryseobacterium piperi isolated from freshwater sediments; however, the biological activity of bont-related genes has not been noted. Additionally, Enterococcus faecium strains have been discovered carrying BoNT-related clusters (BoNT/En). Literature data regarding the heterogeneity of BoNT-producing strains indicate the requirement to reclassify C. botulinum species and other microorganisms able to produce BoNTs or possess botulinum-like gene clusters. This article aims to show the dual nature of Clostridium strains not belonging to the C. botulinum species that are sporadically able to carry bont clusters, which are usually considered saprophytic and even probiotic, and bont-like clusters in microorganisms from other genera. The aim was also to consider the genetic mechanisms of botulinum cluster expression in strains that are considered opportunistic and the microbiological safety aspects associated with their occurrence in the environment. Full article

Avian botulism is a paralytic disease due to the ingestion of botulinum neurotoxins (BoNT) produced by anaerobic, sporigenic bacteria (notably, Clostridium botulinum). Wild waterbirds worldwide are affected with variable recurrence and severity, and organic material decaying in wetland habitats may constitute a suitable substrate for the replication of clostridia strains producing BoNT in conditions of high temperatures and the absence of oxygen. Here, we describe a large outbreak of avian botulism that occurred in the Valle Mandriole protected area of northeastern Italy (VM). After the recovery in late summer of a few duck carcasses that molecularly tested positive for BoNT-producing clostridia, in October 2019, the avian botulism escalation led to a total of 2367 birds being recovered (2158 carcasses and 209 sick birds). Among these, 2365/2367 were waterbirds, with ducks accounting for 91.8% of the total (2173/2367) and green-winged teals representing 93.5% of the ducks. After the quick collection of dead and sick birds (from 4 to 11 October 2019) and the flooding of the VM wetland (from 5 to 12 October 2019), the 2019 botulism emergency apparently ended. Following two water inputs in May and July 2020, only one pooled sample obtained from 16 bird carcasses found that year in VM tested positive for clostridia type C by real-time PCR, whereas, after to the implementation of measures deterring the bird’s presence, new avian botulism cases—due to clostridia type C and C/D, according to molecular and animal-model tests of confirmation—led to the collection of 176 waterbirds (82 carcasses and 94 sick ducks) and 16 waterbirds (9 carcasses and 7 sick ducks) in the summers 2021 and 2022, respectively. In conclusion, the prevention, management, and control of the disease rely on habitat management, the quick and careful collection/removal of animal carcasses, and the regular monitoring and surveillance of live and dead birds. Full article

Avian botulism caused by Clostridium botulinum emerged in 1910, affecting birds across North America, leading to severe outbreaks exacerbated by climate change, decreasing water levels, and inadequate wastewater management. While deadly for birds, its epidemiological impact on humans and other animals remains limited. Despite its significance, understanding and controlling the disease remain challenging. This review delves into the pathogen’s epidemiology in wild bird populations, exploring the transmission, pathogenicity, clinical symptoms, diagnosis and treatment. The disease’s growing concern in wild birds relates to the bacterium’s adaptability and expansive spread, evident through genetic similarities among strains across countries. Outbreaks are influenced by environmental factors such as temperature and soil characteristics. Wild birds inadvertently transmit the bacterium, perpetuating the cycle through carcasses and flies. Some species suffer severely, while others, like scavengers, show resistance. Understanding disease mechanisms, involving potential toxin ingestion or internal production, remains ongoing. Clinical signs vary, affecting diverse bird orders. Diagnostic methods evolve, with treatment success varying among affected populations. Prevention and surveillance take precedence due to treatment challenges, emphasising population-based strategies and preventive measures to manage the widespread presence of C. botulinum. Full article

Clostridium botulinum is the main causative agent of botulism in humans and animals. The ingestion of the botulinum neurotoxin, usually types C and D, has been shown to produce disease (neurological symptoms) in most botulism cases in cattle. We report an outbreak in Southern Sardinia that involved a livestock farm with 120 animals, 39 of which died. The aim of this report is to describe the course of this outbreak and the progression of symptoms up to the death of some animals; we also describe the therapeutic approach applied in this case and the analytical techniques used to diagnose the disease. Finally, we emphasize the importance of promptly proceeding with the sampling of several matrixes when a suspicion of botulism arises. Full article

Botulinum neurotoxins (BoNTs) are emerging as multipurpose therapeutic compounds for the treatment of several different syndromes involving peripheral and central nervous systems, and muscular and musculoskeletal disorders both in human and veterinary medicine. Therefore, the study of BoNTs is rapidly developing and identifying newly produced BoNT variants. Efforts should be made to clarify the biological and pharmacological characteristics of these novel BoNTs as well as the natural ones. The high potential of BoNTs as a therapeutic compound for medical syndromes lies in its ability to reach a specific cell type while bypassing other cells, thus having mild or no side effects. In this paper the recent developments in BoNTs are reviewed with the aim of analyzing the current knowledge on BoNTs’ biological mechanisms of action, immunogenicity, formulations, and therapeutic applications in the veterinary field, highlighting advantages and drawbacks and identifying the gaps to be filled in order to address research priorities. Full article

The diversity of BoNT-producing Clostridia is still a worrying problem for specialists who explore the evolutionary and taxonomic diversity of C. botulinum. It is also a problem for epidemiologists and laboratory staff conducting investigations into foodborne botulism in humans and animals, because their genetic and phenotypic heterogeneity cause complications in choosing the proper analytical tools and in reliably interpreting results. Botulinum neurotoxins (BoNTs) are produced by several bacterial groups that meet all the criteria of distinct species. Despite this, the historical designation of C. botulinum as the one species that produces botulinum toxins is still exploited. New genetic tools such as whole-genome sequencing (WGS) indicate horizontal gene transfer and the occurrence of botulinum gene clusters that are not limited only to Clostridium spp., but also to Gram-negative aerobic species. The literature data regarding the mentioned heterogeneity of BoNT-producing Clostridia indicate the requirement to reclassify C. botulinum species and other microorganisms able to produce BoNTs or possessing botulinum-like gene clusters. The aim of this study was to present the problem of the diversity of BoNT-producing Clostridia over time and new trends toward obtaining a reliable classification of these microorganisms, based on a complex review of the literature. Full article

Botulinum neurotoxins (BoNT) are the most potent toxins in the world. They are produced by a few dozens of strains within several clostridial species. The toxin that they produce can cause botulism, a flaccid paralysis in humans and other animals. With seven established serologically different types and over 40 subtypes, BoNTs are among the most diverse known toxins. The toxin, its structure, its function and its physiological effects on the neural cell and animal hosts along with its diversity have been the subjects of numerous studies. However, many gaps remain in our knowledge about the BoNT toxin and the species that produce them. One of these gaps involves the distribution and extent of variability along the full length of the gene and the protein as well as its domains and subdomains. In this study, we performed an extensive analysis of all of the available 143 unique BoNT-encoding genes and their products, and we investigated their diversity and evolution. Our results indicate that while the nucleotide variability is almost uniformly distributed along the entire length of the gene, the amino acid variability is not. We found that most of the differences were concentrated along the protein’s light chain (LC) domain and especially, the C-terminus of the receptor-binding domain (H_CC). These two regions of the protein are thus identified as the main source of the toxin type differentiation, and consequently, this toxin’s versatility to bind different receptors and their isoforms and act upon different substrates, thus infecting different hosts. Full article

Botulinum neurotoxins (BoNTs) produced by the bacteria Clostridium botulinum are the causative agent of human and animal botulism, a rare but serious and potentially deadly intoxication. Foodborne botulism is caused by the consumption of foods containing BoNTs, which results from contamination of foods with C. botulinum spores and toxin production by the bacteria during growth within the food. Validation of the safety of food products is essential in preventing foodborne botulism, however, limited guidance and standards exist for the selection of strains used in C. botulinum food challenge studies. Sequencing and genomics studies have revealed that C. botulinum is a large, diverse, and polyphyletic species, with physiologic and growth characteristics studied only in a few representatives. Little is known about potential growth competition or effects on toxin production between C. botulinum strains. In this study, we investigated an applied cocktail of ten C. botulinum strains, seven Group I and three Group II. Whole genome SNP alignments revealed that this strain cocktail encompasses the major clades of the Group I and II C. botulinum species. While growth competition appears to exist between several of the strains, the cocktail as a whole resulted in high levels of BoNT production. Full article

Clostridium botulinum is a notorious pathogen that raises health and food safety concerns by producing the potent botulinum neurotoxin and causing botulism, a potentially fatal neuroparalytic disease in humans and animals. Efficient methods for the identification and isolation of C. botulinum are warranted for laboratory diagnostics of botulism and for food safety risk assessment. The cell wall binding domains (CBD) of phage lysins are recognized by their high specificity and affinity to distinct types of bacteria, which makes them promising for the development of diagnostic tools. We previously identified CBO1751, which is the first antibotulinal phage lysin showing a lytic activity against C. botulinum Group I. In this work, we assessed the host specificity of the CBD of CBO1751 and tested its feasibility as a probe for the specific isolation of C. botulinum Group I strains. We show that the CBO1751 CBD specifically binds to C. botulinum Group I sensu lato (including C. sporogenes) strains. We also demonstrate that some C. botulinum Group I strains possess an S-layer, the disruption of which by an acid glycine treatment is required for efficient binding of the CBO1751 CBD to the cells of these strains. We further developed CBO1751 CBD-based methods using flow cytometry and magnetic separation to specifically isolate viable cells of C. botulinum Group I. These methods present potential for applications in diagnostics and risk assessment in order to control the botulism hazard. Full article

Clostridium botulinum and Clostridium tetani are Gram-positive, spore-forming, and anaerobic bacteria that produce the most potent neurotoxins, botulinum toxin (BoNT) and tetanus toxin (TeNT), responsible for flaccid and spastic paralysis, respectively. The main habitat of these toxigenic bacteria is the environment (soil, sediments, cadavers, decayed plants, intestinal content of healthy carrier animals). C. botulinum can grow and produce BoNT in food, leading to food-borne botulism, and in some circumstances, C. botulinum can colonize the intestinal tract and induce infant botulism or adult intestinal toxemia botulism. More rarely, C. botulinum colonizes wounds, whereas tetanus is always a result of wound contamination by C. tetani. The synthesis of neurotoxins is strictly regulated by complex regulatory networks. The highest levels of neurotoxins are produced at the end of the exponential growth and in the early stationary growth phase. Both microorganisms, except C. botulinum E, share an alternative sigma factor, BotR and TetR, respectively, the genes of which are located upstream of the neurotoxin genes. These factors are essential for neurotoxin gene expression. C. botulinum and C. tetani share also a two-component system (TCS) that negatively regulates neurotoxin synthesis, but each microorganism uses additional distinct sets of TCSs. Neurotoxin synthesis is interlocked with the general metabolism, and CodY, a master regulator of metabolism in Gram-positive bacteria, is involved in both clostridial species. The environmental and nutritional factors controlling neurotoxin synthesis are still poorly understood. The transition from amino acid to peptide metabolism seems to be an important factor. Moreover, a small non-coding RNA in C. tetani, and quorum-sensing systems in C. botulinum and possibly in C. tetani, also control toxin synthesis. However, both species use also distinct regulatory pathways; this reflects the adaptation of C. botulinum and C. tetani to different ecological niches. Full article

Botulinum neurotoxins (BoNTs) are produced as protein complexes by bacteria of the genus Clostridium that are Gram-positive, anaerobic and spore forming (Clostridium botulinum, C. butyricum, C. baratii and C. argentinense spp.). BoNTs show a high immunological and genetic diversity. Therefore, fast, precise, and more reliable detection methods are still required to monitor outbreaks and ensure surveillance of botulism. The botulinum toxin field also comprises therapeutic uses, basic research studies and biodefense issues. This review presents currently available detection methods, and new methods offering the potential of enhanced precision and reproducibility. While the immunological methods offer a range of benefits, such as rapid analysis time, reproducibility and high sensitivity, their implementation is subject to the availability of suitable tools and reagents, such as specific antibodies. Currently, the mass spectrometry approach is the most sensitive in vitro method for a rapid detection of active or inactive forms of BoNTs. However, these methods require inter-laboratory validation before they can be more widely implemented in reference laboratories. In addition, these surrogate in vitro models also require full validation before they can be used as replacement bioassays of potency. Cell-based assays using neuronal cells in culture recapitulate all functional steps of toxin activity, but are still at various stages of development; they are not yet sufficiently robust, due to high batch-to-batch cell variability. Cell-based assays have a strong potential to replace the mouse bioassay (MBA) in terms of BoNT potency determination in pharmaceutical formulations; they can also help to identify suitable inhibitors while reducing the number of animals used. However, the development of safe countermeasures still requires the use of in vivo studies to complement in vitro immunological or cell-based approaches. Full article

The clostridial diseases of horses can be divided into three major groups: enteric/enterotoxic, histotoxic, and neurotoxic. The main enteric/enterotoxic diseases include those produced by Clostridium perfringens type C and Clostridioides difficile, both of which are characterized by enterocolitis. The main histotoxic diseases are gas gangrene, Tyzzer disease, and infectious necrotic hepatitis. Gas gangrene is produced by one or more of the following microorganisms: C. perfringens type A, Clostridium septicum, Paeniclostridium sordellii, and Clostridium novyi type A, and it is characterized by necrotizing cellulitis and/or myositis. Tyzzer disease is produced by Clostridium piliforme and is mainly characterized by multifocal necrotizing hepatitis. Infectious necrotic hepatitis is produced by Clostridium novyi type B and is characterized by focal necrotizing hepatitis. The main neurotoxic clostridial diseases are tetanus and botulism, which are produced by Clostridium tetani and Clostridium botulinum, respectively. Tetanus is characterized by spastic paralysis and botulism by flaccid paralysis. Neither disease present with specific gross or microscopic lesions. The pathogenesis of clostridial diseases involves the production of toxins. Confirming a diagnosis of some of the clostridial diseases of horses is sometimes difficult, mainly because some agents can be present in tissues of normal animals. This paper reviews the main clostridial diseases of horses. Full article