Paretic syndrome in gulls from southern Portugal: searching for the causative agent

Between 2010 and 2019, 2.432 gulls (Larus michahellis and Larus fuscus) with paretic syndrome were received at RIAS Wildlife Rehabilitation and Research Centre. The clinical signs included weakness, anorexia, paralysis, diarrhoea (flaccid cloacae), dyspnoea and, in some cases, death. Several biotic contaminants are among the potential cause of this syndrome: marine biotoxins, Clostridium botulinum, cyanotoxins and virus. This presentation compiles the results of the Clostridium botulinum and marine biotoxins analysis conducted at the French National Reference Centre for anaerobic bacteria and botulism, Pasteur Institute (Paris) and the Vigo Centre of the Spanish Oceanographic Institute, respectively. C. botulinum analyses were conducted in livers and intestines from 5 gulls with paretic syndrome symptoms admitted at RIAS Wildlife Rehabilitation and Research Centre. Samples were pooled in two groups according to the tissue and analysed by targeted Real Time Polymerase Chain Reaction (PCR) on neurotoxin genes after sample enrichment culture under anaerobic conditions. The presence of botulinum toxin was confirmed by a lethality test on mice (mouse bioassay). Mice were intraperitoneally injected with filtered supernatant of the culture. Paralytic shellfish toxins (PSTs) were analysed by Liquid Chromatography with Fluorescence Detection and Post-column Oxidation in samples from ten gull kidneys and in the cloacae contents from another gull. Domoic acid (DA) analysis was conducted following a procedure that involved a methanolic extraction and analysis by Liquid Chromatography coupled to High Resolution Mass Spectrometry. DA was analysed in twenty three gull samples: ten livers, ten intestines and three cloacae contents. PSTs and DA were not detected in any of the samples tested. Results obtained so far point to C. botulimum type C/D as the causative agent of the paretic syndrome in gulls

Closing Clostridium botulinum Group III Genomes Using Long-Read Sequencing

Clostridium botulinum group III is the anaerobic Gram-positive bacterium producing the deadly neurotoxin responsible for animal botulism. Here, we used long-read sequencing to produce four complete genomes from Clostridium botulinum group III neurotoxin types C, D, C/D, and D/C. The protocol for obtaining high-molecular-weight DNA from C. botulinum group III is described.Peer reviewe

Production and Characterisation of a Neutralising Chimeric Antibody against Botulinum Neurotoxin A

Botulinum neurotoxins, produced by Clostridium botulinum bacteria, are the causative agent of botulism. This disease only affects a few hundred people each year, thus ranking it among the orphan diseases. However, botulinum toxin type A (BoNT/A) is the most potent toxin known to man. Due to their potency and ease of production, these toxins were classified by the Centers for Disease Control and Prevention (CDC) as Category A biothreat agents. For several biothreat agents, like BoNT/A, passive immunotherapy remains the only possible effective treatment allowing in vivo neutralization, despite possible major side effects. Recently, several mouse monoclonal antibodies directed against a recombinant fragment of BoNT/A were produced in our laboratory and most efficiently neutralised the neurotoxin. In the present work, the most powerful one, TA12, was selected for chimerisation. The variable regions of this antibody were thus cloned and fused with the constant counterparts of human IgG1 (kappa light and gamma 1 heavy chains). Chimeric antibody production was evaluated in mammalian myeloma cells (SP2/0-Ag14) and insect cells (Sf9). After purifying the recombinant antibody by affinity chromatography, the biochemical properties of chimeric and mouse antibody were compared. Both have the same very low affinity constant (close to 10 pM) and the chimeric antibody exhibited a similar capacity to its parent counterpart in neutralising the toxin in vivo. Its strong affinity and high neutralising potency make this chimeric antibody interesting for immunotherapy treatment in humans in cases of poisoning, particularly as there is a probable limitation of the immunological side effects observed with classical polyclonal antisera from heterologous species

The European AntibotABE Framework Program and Its Update: Development of Innovative Botulinum Antibodies

The goal of the AntiBotABE Program was the development of recombinant antibodies that neutralize botulinum neurotoxins (BoNT) A, B and E. These serotypes are lethal and responsible for most human botulinum cases. To improve therapeutic efficacy, the heavy and light chains (HC and LC) of the three BoNT serotypes were targeted to achieve a synergistic effect (oligoclonal antibodies). For antibody isolation, macaques were immunized with the recombinant and non-toxic BoNT/A, B or E, HC or LC, followed by the generation of immune phage-display libraries. Antibodies were selected from these libraries against the holotoxin and further analyzed in in vitro and ex vivo assays. For each library, the best ex vivo neutralizing antibody fragments were germline-humanized and expressed as immunoglobulin G (IgGs). The IgGs were tested in vivo, in a standardized model of protection, and challenged with toxins obtained from collections of Clostridium strains. Protective antibody combinations against BoNT/A and BoNT/B were evidenced and for BoNT/E, the anti-LC antibody alone was found highly protective. The combination of these five antibodies as an oligoclonal antibody cocktail can be clinically and regulatorily developed while their high “humanness” predicts a high tolerance in humans.Peer reviewe

Development of Human-Like scFv-Fc Neutralizing Botulinum Neurotoxin E

Background Botulinum neurotoxins (BoNTs) are considered to be the most toxic substances known on earth and are responsible for human botulism, a life-threatening disease characterized by flaccid muscle paralysis that occurs naturally by food-poisoning or colonization of the gastrointestinal tract by BoNT-producing clostridia. BoNTs have been classified as category A agent by the Centers of Disease Control and Prevention (CDC) and are listed among the six agents with the highest risk to be used as bioweapons. Neutralizing antibodies are required for the development of effective anti-botulism therapies to deal with the potential risk of exposure. Results In this study, a macaque (Macaca fascicularis) was immunized with recombinant light chain of BoNT/E3 and an immune phage display library was constructed. After a multi-step panning, several antibody fragments (scFv, single chain fragment variable) with nanomolar affinities were isolated, that inhibited the endopeptidase activity of pure BoNT/E3 in vitro by targeting its light chain. Furthermore, three scFv were confirmed to neutralize BoNT/E3 induced paralysis in an ex vivo mouse phrenic nerve-hemidiaphragm assay. The most effective neutralization (20LD50/mL, BoNT/E3) was observed with scFv ELC18, with a minimum neutralizing concentration at 0.3 nM. Furthermore, ELC18 was highly effective in vivo when administered as an scFv-Fc construct. Complete protection of 1LD50 BoNT/E3 was observed with 1.6 ng/dose in the mouse flaccid paralysis assay. Conclusion These scFv-Fcs antibodies are the first recombinant antibodies neutralizing BoNT/E by targeting its light chain. The human-like nature of the isolated antibodies is predicting a good tolerance for further clinical development.Peer reviewe

Historical Perspectives and Guidelines for Botulinum Neurotoxin Subtype Nomenclature

Botulinum neurotoxins are diverse proteins. They are currently represented by at least seven serotypes and more than 40 subtypes. New clostridial strains that produce novel neurotoxin variants are being identified with increasing frequency, which presents challenges when organizing the nomenclature surrounding these neurotoxins. Worldwide, researchers are faced with the possibility that toxins having identical sequences may be given different designations or novel toxins having unique sequences may be given the same designations on publication. In order to minimize these problems, an ad hoc committee consisting of over 20 researchers in the field of botulinum neurotoxin research was convened to discuss the clarification of the issues involved in botulinum neurotoxin nomenclature. This publication presents a historical overview of the issues and provides guidelines for botulinum neurotoxin subtype nomenclature in the future.Peer reviewe

Characterization of Botulinum Neurotoxin Type A Neutralizing Monoclonal Antibodies and Influence of Their Half-Lives on Therapeutic Activity

Botulinum toxins, i.e. BoNT/A to/G, include the most toxic substances known. Since botulism is a potentially fatal neuroparalytic disease with possible use as a biowarfare weapon (Centers for Disease Control and Prevention category A bioterrorism agent), intensive efforts are being made to develop vaccines or neutralizing antibodies. The use of active fragments from non-human immunoglobulins (F(ab')2, Fab', scFv), chemically modified or not, may avoid side effects, but also largely modify the in vivo half-life and effectiveness of these reagents. We evaluated the neutralizing activity of several monoclonal anti-BoNT/A antibodies (mAbs). F(ab')2 fragments, native or treated with polyethyleneglycol (PEG), were prepared from selected mAbs to determine their half-life and neutralizing activity as compared with the initial mAbs. We compared the protective efficiency of the different biochemical forms of anti-toxin mAbs providing the same neutralizing activity. Among fourteen tested mAbs, twelve exhibited neutralizing activity. Fragments from two of the best mAbs (TA12 and TA17), recognizing different epitopes, were produced. These two mAbs neutralized the A1 subtype of the toxin more efficiently than the A2 or A3 subtypes. Since mAb TA12 and its fragments both exhibited the greatest neutralizing activity, they were further evaluated in the therapeutic experiments. These showed that, in a mouse model, a 2- to 4-h interval between toxin and antitoxin injection allows the treatment to remain effective, but also suggested an absence of correlation between the half-life of the antitoxins and the length of time before treatment after botulinum toxin A contamination. These experiments demonstrate that PEG treatment has a strong impact on the half-life of the fragments, without affecting the effectiveness of neutralization, which was maintained after preparation of the fragments. These reagents may be useful for rapid treatment after botulinum toxin A contamination

Clostridium botulinum type C, D, C/D, and D/C: An update

Clostridium botulinum is the main causative agent of botulism, a neurological disease encountered in humans as well as animals. Nine types of botulinum neurotoxins (BoNTs) have been described so far. Amongst these “toxinotypes,” the A, the B and E are the most frequently encountered in humans while the C, D, C/D and D/C are mostly affecting domestic and wild birds as well as cattle. In France for instance, many cases and outbreaks are reported in these animal species every year. However, underestimation is very likely at least for avifauna species where the detection of dead animals can be challenging. Knowledge about BoNTs C, D, C/D, and D/C and the diseases they cause in animals and humans is still scarce and unclear. Specifically, the potential role of animal botulism outbreaks in cattle and poultry as a source of human illness needs to be further assessed. In this narrative review, we present the current knowledge about toxinotypes C, D, C/D, and D/C in cattle and poultry with, amongst various other aspects, their epidemiological cycles. We also discuss the zoonotic potential of these toxinotypes and some possible ways of risk mitigation. An adapted and effective management of botulism outbreaks in livestock also requires a better understanding of these less common and known toxinotypes

Draft Genome Sequence of Clostridium botulinum Strain 277-00 Type B2

International audienceWe report the draft genome sequence of Clostridium botulinum strain 277-00, which encodes a botulinum neurotoxin B2 associated with a ha gene locus. Strain 277-00 was isolated from a cheese responsible for an outbreak of botulism in Iran in 1997. This strain is closed to the bivalent B2/FA strain IBCA10-7060

Botulism and Tetanus

DOI de la monographie : 10.1007/978-3-642-30144-5International audienceBotulism and tetanus are two severe neurological diseases in man and animals. While botulism is characterized by a descendant flaccid paralysis, tetanus consists in spastic paralysis. In the severe forms of both diseases, death occurs by respiratory distress. Botulism and tetanus are caused by neurotoxins, botulinum neurotoxin (BoNT), and tetanus toxin (TeNT), respectively, which are produced by anaerobic sporulating bacteria, Clostridium botulinum and Clostridium tetani, respectively. In contrast to C. tetani, which forms a homogeneous bacterial species, BoNT-producing Clostridia are divided into several bacterial species and groups. These Clostridia are widely distributed in the environment, including food notably for C. botulinum, where they can survive during long periods in the sporulating forms. BoNTs and TeNT share a common structural organization consisting in a light (L) chain (about 50 kDa) linked by a disulfide bridge to the heavy (H) chain (about 100 kDa). Only a unique TeNT is known, while BoNTs encompass seven toxinotypes (A to G, BoNT/A, B, and E mainly involved in human botulism, and BoNT/C and D mainly responsible for animal botulism), which are subdivided into several subtypes according to amino acid sequence variations. H chain, which contains a C-terminal receptor-binding domain and an N-terminal translocation domain, delivers the L chain into target neurons. BoNTs target the motoneuron endings or neuromuscular junctions, and TeNT is transported to central inhibitory interneurons through a retrograde axonal pathway along motoneurons. Both BoNT and TeNT block the release of neurotransmitter by an L chain-mediated proteolytic cleavage of SNARE proteins (synaptobrevin, SNAP25, or syntaxin) which are involved in the neuroexocytosis process. Blockage of acetylcholine release at the neuromuscular junctions by BoNTs induces a flaccid paralysis, whereas TeNT-dependent inhibition of glycine or GABA exocytosis in inhibitory interneurons results in spastic paralysis. Botulism is mainly acquired by ingestion of preformed BoNT in food, but it may also occur subsequently to intestinal or wound colonization by C. botulinum. Tetanus essentially results from a wound contamination by C. tetani. BoNT/A is the most potent toxin with a long activity duration in neurons, and it is also a therapeutic agent widely used to treat hypercholinergic diseases including localized muscle spasticity, dystonia, autonomic dysfunctions (hyperhidrosis, hypersalivation), and also pain such as migraine headaches