Accidental and intended Ricinus communis intoxications in humans and animals have been known for centuries but the causative agent remained elusive until 1888 when Stillmark attributed the toxicity to the lectin ricin. Ricinus communis is grown worldwide on an industrial scale for the production of castor oil. As by-product in castor oil production ricin is mass produced above 1 million tons per year. On the basis of its availability, toxicity, ease of preparation and the current lack of medical countermeasures, ricin has gained attention as potential biological warfare agent. The seeds also contain the less toxic, but highly homologous Ricinus communis agglutinin and the alkaloid ricinine, and especially the latter can be used to track intoxications. After oil extraction and detoxification, the defatted press cake is used as organic fertilizer and as low-value feed. In this context there have been sporadic reports from different countries describing animal intoxications after uptake of obviously insufficiently detoxified fertilizer. Observations in Germany over several years, however, have led us to speculate that the detoxification process is not always performed thoroughly and controlled, calling for international regulations which clearly state a ricin threshold in fertilizer. In this review we summarize knowledge on intended and unintended poisoning with ricin or castor seeds both in humans and animals, with a particular emphasis on intoxications due to improperly detoxified castor bean meal and forensic analysis.

Botulinum neurotoxins are produced by the anaerobic bacteriumClostridium botulinumand are divided into seven distinct serotypes (A to G) known to cause botulism in animals and humans. In this study, a multiplexed quantitative real-time PCR assay for the simultaneous detection of the human pathogenicC. botulinumserotypes A, B, E, and F was developed. Based on the TaqMan chemistry, we used five individual primer-probe sets within one PCR, combining both minor groove binder- and locked nucleic acid-containing probes. Each hydrolysis probe was individually labeled with distinguishable fluorochromes, thus enabling discrimination between the serotypes A, B, E, and F. To avoid false-negative results, we designed an internal amplification control, which was simultaneously amplified with the four target genes, thus yielding a pentaplexed PCR approach with 95% detection probabilities between 7 and 287 genome equivalents per PCR. In addition, we developed six individual singleplex real-time PCR assays based on the TaqMan chemistry for the detection of theC. botulinumserotypes A, B, C, D, E, and F. Upon analysis of 42C. botulinumand 57 non-C. botulinumstrains, the singleplex and multiplex PCR assays showed an excellent specificity. Using spiked food samples we were able to detect between 10³and 10⁵CFU/ml, respectively. Furthermore, we were able to detect C.botulinumin samples from several cases of botulism in Germany. Overall, the pentaplexed assay showed high sensitivity and specificity and allowed for the simultaneous screening and differentiation of specimens forC. botulinumA, B, E, and F.

The detection and identification of botulinum neurotoxins (BoNT) is complex due to the existence of seven serotypes, derived mosaic toxins and more than 40 subtypes. Expert laboratories currently use different technical approaches to detect, identify and quantify BoNT, but due to the lack of (certified) reference materials, analytical results can hardly be compared. In this study, the six BoNT/A1–F1 prototypes were successfully produced by recombinant techniques, facilitating handling, as well as improving purity, yield, reproducibility and biosafety. All six BoNTs were quantitatively nicked into active di-chain toxins linked by a disulfide bridge. The materials were thoroughly characterized with respect to purity, identity, protein concentration, catalytic and biological activities. For BoNT/A₁, B₁ and E₁, serotypes pathogenic to humans, the catalytic activity and the precise protein concentration were determined by Endopep-mass spectrometry and validated amino acid analysis, respectively. In addition, BoNT/A₁, B₁, E₁ and F₁ were successfully detected by immunological assays, unambiguously identified by mass spectrometric-based methods, and their specific activities were assigned by the mouse LD₅₀ bioassay. The potencies of all six BoNT/A1–F1 were quantified by the ex vivo mouse phrenic nerve hemidiaphragm assay, allowing a direct comparison. In conclusion, highly pure recombinant BoNT reference materials were produced, thoroughly characterized and employed as spiking material in a worldwide BoNT proficiency test organized by the EQuATox consortium.

The exceptional toxicity of botulinum neurotoxins (BoNTs) is mediated by high avidity binding to complex polysialogangliosides and intraluminal segments of synaptic vesicle proteins embedded in the presynaptic membrane. One peculiarity is an exposed hydrophobic loop in the toxin’s cell binding domain H_C, which is located between the ganglioside- and protein receptor-binding sites, and that is particularly pronounced in the serotypes BoNT/B, DC, and G sharing synaptotagmin as protein receptor. Here, we provide evidence that this H_C loop is a critical component of their tripartite receptor recognition complex. Binding to nanodisc-embedded receptors and toxicity were virtually abolished in BoNT mutants lacking residues at the tip of the H_C loop. Surface plasmon resonance experiments revealed that only insertion of the H_C loop into the lipid-bilayer compensates for the entropic penalty inflicted by the dual-receptor binding. Our results represent a new paradigm of how BoNT/B, DC, and G employ ternary interactions with a protein, ganglioside, and lipids to mediate their extraordinary neurotoxicity. Botulinum neurotoxins are Janus-faced molecules: due to their exquisite toxicity, botulinum neurotoxins are considered as biological weapons, but they are also highly effective medicines for numerous neurological indications. However, what mediates their exquisite toxicity? The exclusive binding to neurons and the subsequent paralysis cuts off the host’s communication networks. The neurospecific binding is ensured by anchoring to two receptor molecules both embedded in the membrane: a complex ganglioside and a synaptic vesicle protein. Here, we reveal a third interaction between a hydrophobic so-called H_C loop protruding from the surface of the serotypes BoNT/B, DC, and G into the lipid membrane. Only this H_C loop ensures their high-affinity binding to the neuronal receptors also at physiological temperature (37°C). Hereby, BoNT/B, DC, and G prevent untimely dissociation prior to uptake into the neuron. Therefore, our study provides the mechanistic basis for the development of inhibitors to combat botulism, but it also has implications for engineering toxin—membrane interactions yielding optimized BoNT-based therapeutics to treat neuromuscular dysfunctions successfully. Intriguingly, a broadly neutralizing anti-HIV-1 antibody shares a similar strategy, emphasizing the general relevance of our results for host—pathogen interactions.

In March 2023, 34 associated cases of iatrogenic botulism were detected in Germany (30 cases), Switzerland (two cases), Austria (one case), and France (one case). An alert was rapidly disseminated via European Union networks and communication platforms (Food- and Waterborne Diseases and Zoonoses Network, EpiPulse, Early Warning and Response System) and the International Health Regulation mechanism; the outbreak was investigated in a European collaboration. We traced sources of the botulism outbreak to treatment of weight loss in Türkiye, involving intragastric injections of botulinum neurotoxin. Cases were traced using a list of patients who had received this treatment. Laboratory investigations of the first 12 German cases confirmed nine cases. The application of innovative and highly sensitive endopeptidase assays was necessary to detect minute traces of botulinum neurotoxin in patient sera. The botulism notification requirement for physicians was essential to detect this outbreak in Germany. The surveillance case definition of botulism should be revisited and inclusion of cases of iatrogenic botulism should be considered as these cases might lack standard laboratory confirmation yet warrant public health action. Any potential risks associated with the use of botulinum neurotoxins in medical procedures need to be carefully balanced with the expected benefits of the procedure.

Botulinum neurotoxins (BoNTs) cause the life-threatening neurological illness botulism in humans and animals and are divided into seven serotypes (BoNT/A–G), of which serotypes A, B, E, and F cause the disease in humans. BoNTs are classified as “category A” bioterrorism threat agents and are relevant in the context of the Biological Weapons Convention. An international proficiency test (PT) was conducted to evaluate detection, quantification and discrimination capabilities of 23 expert laboratories from the health, food and security areas. Here we describe three immunological strategies that proved to be successful for the detection and quantification of BoNT/A, B, and E considering the restricted sample volume (1 mL) distributed. To analyze the samples qualitatively and quantitatively, the first strategy was based on sensitive immunoenzymatic and immunochromatographic assays for fast qualitative and quantitative analyses. In the second approach, a bead-based suspension array was used for screening followed by conventional ELISA for quantification. In the third approach, an ELISA plate format assay was used for serotype specific immunodetection of BoNT-cleaved substrates, detecting the activity of the light chain, rather than the toxin protein. The results provide guidance for further steps in quality assurance and highlight problems to address in the future.