Machine learning-based typing of Clostridium botulinum group III by FT-IR spectroscopy Ilenia Drigo, Angela Guolo, Alessia Rizzardi, Miriam Cordovana, Manuel Garbuio, Elena Tonon, Marco Vedana, Luca Zandonà, Luca Bano Microbiology Spectrum, 2026 This study aimed to investigate the utility of Fourier-Transform Infrared Spectroscopy (FT-IRS) for differentiating Clostridium botulinum ( C. botulinum ) based on its botulinum neurotoxin (BoNT)-encoding gene type and its potential as an epidemiological tool for investigating botulism outbreaks. A total of 110 botulinum neurotoxin-producing clostridia (BNPC) strains, including reference, animal isolates, and human outbreak strains, were analyzed in four replicates using the IR Biotyper system (IRBT). Samples preparation was carried out according to the manufacturer’s instructions. Similarity analysis was performed by hierarchical cluster analysis (HCA), principal component analysis (PCA), and linear discriminant analysis (LDA). The artificial intelligence capabilities of the IRBT software were applied to develop a classifier for C. botulinum differentiation at toxin-serotype or subtype level. HCA, PCA, and LDA showed a good clustering of strains belonging to the same type. In accordance with the lineages evidenced in whole-genome sequencing (WGS) studies, types A, B, and F BNPC appeared clearly separated from types C, CD, DC, and D. Considering only C, CD, DC, and D types, the highest discriminatory power is achieved in the wavenumber range 1,800–1,500 cm −1 . Four different clusters were detected. Support vector machine algorithm with linear kernel (Linear SVM) showed the highest accuracy of discrimination at the BoNT type level with an accuracy of 97%. Although these preliminary results need to be confirmed with a higher number of strains, the IRBT system proved to be a very promising, user-friendly, and cost-effective tool for C. botulinum typing, and the application of machine learning algorithms represents a novel approach for BNPC typing. IMPORTANCE Botulism outbreaks represent a significant threat to public and animal health. Rapid and accurate typing methods are essential for effective epidemiological investigations, source tracing, and the implementation of appropriate control measures. Current methods for botulinum neurotoxin serotyping are often time-consuming, expensive, and require specialized expertise. Our research demonstrated that FT-IRS, a rapid, user-friendly, and cost-effective technique already well established in microbiology for broader bacterial characterization, can be successfully adapted for this crucial task. The use of a commercially available system like the IRBT significantly enhances the potential for widespread adoption of this methodology in routine diagnostics and surveillance.
Intra-farm dynamic of environmental conditions in commercial rabbitries with respiratory disorders using an innovative IoT device Manuel Garbuio, Benedetta Cordioli, Elisabetta Riva, Luca Bano, Giorgio Provolo Smart Agricultural Technology, 2026 • Use of multifunctional real-time IoT devices in rabbitries is a valuable tool for farmers’ decision-making, helping to mitigate the impact of environmental conditions on animal health. • Farms with recurrent problems of respiratory disease sustained by Pasteurella multocida may have critical environmental predisposing factors. • Key environmental parameters have a non-homogeneous distribution inside problematic rabbitries. • Dustiness, sound pressure and sulphuric acid are not a problem in North-Italy rabbitries. The microclimate inside rabbitries is a well-established predisposing factor for respiratory diseases, influencing animal growth and health. This study investigated the fluctuations of key environmental parameters in fattening rabbitries with and without respiratory disorders. To this purpose, six multifunctional IoT (Internet of Things) devices were placed at animal level in different locations within each barn. These devices can measure continuously various parameters, including temperature, relative humidity, light intensity, sound pressure, gas concentrations (ammonia, sulphuric acid, carbon dioxide) and particulate levels. Measurements were collected for 7 to 10 days in each farm, with external conditions simultaneously recorded from local meteorological stations. Considering the optimal welfare values, the continuous monitoring system provided valuable information regarding significant deviations of some parameters, irrespective of the external conditions. The results highlighted that temperatures were consistently above 20 °C for 51%, 89%, and 100% of the time in three out of four problematic farms, whereas relative humidity was below the suggested value of 60% in all of them for 30% to 89% of the time. Moreover, multi-site measurements within the same building revealed a heterogeneous distribution of the parameters investigated. Differences in ammonia concentrations up to 11 ppm have been found in the same barn, indicating a low uniformity of air quality. Dustiness, sound pressure, and sulphuric acid concentrations never reached levels of concern. The findings demonstrate the utility of this real-time evaluation method as a valuable tool for farmers' decision-making, specifically in efforts to mitigate the adverse impact of environmental conditions on animal health.
Botulism Caroline Le Maréchal, Fabrizio Anniballi, Luca Bano, Martin B. Dorner, Miriam Koene, Christelle Mazuet, Christian Seyboldt, Tom Van Nieuwenhuysen Clostridial Diseases of Animals, 2026 Botulism is a severe, flaccid neuroparalytic disease of humans and animals. The disease affects most mammals, birds, and fish. Animal botulism is a major environmental and economic concern, mainly because of the high mortality rate during outbreaks; it is considered to be a zoonosis, as it may be a source of food-borne botulism for humans. It is also considered a potential bioterrorism threat. Botulism can be caused by one of eight different neuroparalytic toxin serotypes (A to H), and it is characterized by the absence of specific gross or microscopic lesions. A presumptive diagnosis of animal botulism is based on clinical signs and ruling out other clinically similar diseases. Confirmation is based on detection of the botulinum toxin in animal samples, feed, and/or water, for which the mouse bioassay is still the gold standard. Several in vitro methods are currently being validated. Prevention of animal botulism can be achieved by proper management and vaccination. Treatment is usually not successful in the advanced stages of the disease, and euthanasia is frequently advised. For mammalian species, antitoxin can be administered to affected animals, but this has to be done when the toxin is circulating before it is bound to the neuromuscular junction or when a progressive toxico-infection form is suspected or in cohort animals.
Experimental model for the reproduction of necrotic enteritis in turkeys using different Clostridium perfringens strains B. Cordioli, M. Vereecken, I. Drigo, M. Garbuio, C. Zanardello, L. Gagliazzo, L. Palazzolo, L. Zandonà, A. Rizzardi, M. De Gussem, K. De Gussem, L. Bano Avian Pathology, 2026 Necrotic enteritis (NE) is a disease affecting poultry caused by virulence factors, primarily toxins, produced by Clostridium perfringens (Cp). Despite NE is well documented in turkey flocks, only one study describes its pathogenesis suggesting a protocol for the induction of the disease. The aim of this study was setting up a challenge model for the experimental reproduction of NE in turkey, in line with clinical signs currently observed in the field.To this purpose, the disease was induced based on the administration of some well-known predisposing factors for chicken (sudden switch to a high protein diet, coccidia) and challenging the birds with Cp strains with different genetic virulence factors isolated from chicken and turkey affected by NE. The predisposing factors were administered to 3 experimental groups (T02, T03, T04). In addition, groups T03 and T04 were challenged with Cp isolated from chicken or turkey, respectively. Group T01 served as negative control. Clinical signs, gross pathology and histopathological lesions were scored and compared between groups.The challenge with the chicken Cp strain induced clinical manifestations of NE (80% mortality) and reproduced lesions both macroscopically and microscopically. In groups challenged with Cp, gross pathology revealed more severe lesions (p<0.001) when compared to the group challenged with the predisposing factors only. Histopathologically, those findings were confirmed only in group T03 in the jejunum (p <0.001).This experimental model will be useful to test the efficacy of control/preventive measures and to better understand the virulence factors involved in the pathogenesis of NE in turkey.
Enteric botulinum neurotoxins facilitate infection by Salmonella and Shigella Science Advances, 2026
Unusual Epidemic of Tyzzer’s Disease in Commercial Rabbit Breeders: Clinical, Pathological, and Therapeutic Observations Benedetta Cordioli, Manuel Garbuio, Luca Palazzolo, Francesco Dorigo, Luca Zandonà, Laura Viel, Claudia Zanardello, Luca Bano Animals, 2025 Clostridium piliforme (Cp) is a pleomorphic spore-forming obligate intracellular bacterium and the causative agent of Tyzzer’s disease. The condition affects multiple species, including rabbits, in which the disease is sporadic in recently weaned animals. This report details a case of disease caused by Cp observed exclusively in breeding rabbits of a commercial farm. The clinical manifestations were a higher mortality rate in does and late-gestation abortions. We performed necropsy and further microbiological, parasitological and histopathological analyses. Anatomopathological lesions were suggestive of Tyzzer’s disease and the presence of Cp was confirmed by PCR. Parasitological analysis tested negative and standard bacteriological examination of intestines revealed a high load of Escherichia coli and Clostridium perfringens, which were considered secondary pathogens. Chlamydophila sp. and Toxoplasma gondii infections were excluded by PCR as causative agents of abortions. Moreover, in the months following the diagnosed outbreak, episodes of subcutaneous edema occurred in multiple does and young breeders born after the resolution of the epidemic. The constant reduction in the use of antimicrobials in recent years could make some neglected diseases emerge again. Therefore, it is crucial to suspect such uncommon pathologies in commercial rabbitries to properly manage them on farms.
Isolation of Clostridium botulinum type C from a wound in a pig Benedetta Cordioli, Manuel Garbuio, Eliana Schiavon, Alessia Rizzardi, Angela Guolo, Serena Genovese, Ilenia Drigo, Luca Bano Journal of Veterinary Diagnostic Investigation, 2025 We report here the isolation of Clostridium botulinum type C from the medial muscles of the thigh of a gestating gilt with a claw wound and without evidence of septicemia. The pig died with paralytic signs, consistent with wound botulism, similar to episodes in humans.
Effectiveness of a Bivalent Recombinant Vaccine on the Production of Neutralizing Antibodies Against BoNT/C, BoNT/D, BoNT/CD e BoNT/DC in Bovines Ilenia Drigo, Luca Zandonà, Elena Tonon, Katia Capello, Luca Bano Vaccines, 2025 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.
Calves as Main Reservoir of Antibiotic Resistance Genes in Dairy Farms Barbara Salerno, Matteo Cornaggia, Raffaella Sabatino, Andrea Di Cesare, Maddalena Furlan, Lisa Barco, Massimiliano Orsini, Benedetta Cordioli, Claudio Mantovani, Luca Bano, Carmen Losasso Frontiers in Public Health, 2022
Genome sequence of the fish brain bacterium Clostridium tarantellae Luca Bano, Matthias Kiel, Gabriele Sales, Andrew C. Doxey, Michael J. Mansfield, Haleluya T. Wami, Marco Schiavone, Ornella Rossetto, Marco Pirazzini, Ulrich Dobrindt, Cesare Montecucco Microbiology Resource Announcements, 2020
Investigation of Clostridium botulinum group III's mobilome content Cédric Woudstra, Caroline Le Maréchal, Rozenn Souillard, Fabrizio Anniballi, Bruna Auricchio, Luca Bano, Marie-Hélène Bayon-Auboyer, Miriam Koene, Isabelle Mermoud, Roseane B. Brito, Francisco C.F. Lobato, Rodrigo O.S. Silva, Martin B. Dorner, Patrick Fach Anaerobe, 2018
Characterization of Pasteurella multocida involved in rabbit infections Francesca Romana Massacci, Chiara Francesca Magistrali, Lucilla Cucco, Ludovica Curcio, Luca Bano, PierMario Mangili, Eleonora Scoccia, Magne Bisgaard, Bent Aalbæk, Henrik Christensen Veterinary Microbiology, 2018
The workshop on animal botulism in europe Hanna Skarin, Annica Tevell Åberg, Cédric Woudstra, Trine Hansen, Charlotta Löfström, Miriam Koene, Luca Bano, Mikael Hedeland, Fabrizio Anniballi, Dario De Medici, Eva Olsson Engvall Biosecurity and Bioterrorism, 2013
Animal botulism outcomes in the ani bio threat project Cédric Woudstra, Annica Tevell Åberg, Hanna Skarin, Fabrizio Anniballi, Dario De Medici, Luca Bano, Miriam Koene, Charlotta Löfström, Trine Hansen, Mikael Hedeland, Patrick Fach Biosecurity and Bioterrorism, 2013
Recurrent detection of Histophilus somni in the genital tract of dairy cattle with reproductive failures in Italy Large Animal Review, 2011
Usutu virus in ITALY: An emergence or a silent infection? Giovanni Savini, Federica Monaco, Calogero Terregino, Annapia Di Gennaro, Luca Bano, Chiara Pinoni, Roberta De Nardi, Paolo Bonilauri, Monica Pecorari, Luigina Di Gialleonardo, Lebana Bonfanti, Andrea Polci, Paolo Calistri, Rossella Lelli Veterinary Microbiology, 2011