Evaluation of long-term immune response in cattle to botulism using a recombinant E. coli bacterin formulated with Montanide™ ISA 50 and aluminum hydroxide adjuvants.

Botulism is a severe disease caused by potent botulinum neurotoxins (BoNTs) produced by Clostridium botulinum. This disease is associated with high-lethality outbreaks in cattle, which have been linked to the ingestion of preformed BoNT serotypes C and D, emphasizing the need for effective vaccines. The potency of current commercial toxoids (formaldehyde-inactivated BoNTs) is assured through tests in guinea pigs according to government regulatory guidelines, but their short-term immunity raises concerns. Recombinant vaccines containing the receptor-binding domain have demonstrated potential for eliciting robust protective immunity. Previous studies have demonstrated the safety and effectiveness of recombinant E. coli bacterin, eliciting high titers of neutralizing antibodies against C. botulinum and C. perfringens in target animal species. In this study, neutralizing antibody titers in cattle and the long-term immune response against BoNT/C and D were used to assess the efficacy of the oil-based adjuvant compared with that of the aluminum hydroxide adjuvant in cattle. The vaccine formulation containing Montanide™ ISA 50 yielded significantly higher titers of neutralizing antibody against BoNT/C and D (8.64 IU/mL and 9.6 IU/mL, respectively) and induced an immune response that lasted longer than the response induced by aluminum, extending between 30 and 60 days. This approach represents a straightforward, cost-effective strategy for recombinant E. coli bacterin, enhancing both the magnitude and duration of the immune response to botulism.

Botulism in Two Juvenile Wild Red Foxes (Vulpes vulpes) in Somerset, England, UK.

Two juvenile red foxes (Vulpes vulpes) were euthanased because of severe nervous signs and paralysis. Detailed postmortem examinations were carried out with bacteriology, histology, and Clostridium botulinum toxin screening, which confirmed botulism as the cause of the clinical signs.

Standardization of the Japanese National Standard, Equine Botulinum Antitoxin Type A, and Factors Affecting Standardization.

Equine botulinum antitoxin is one of the most popular countermeasures for human botulism. The unitage of the antitoxin product is defined according to national minimum requirement or pharmacopoeia in each country by referring to national standard antitoxins for four types (A, B, E, and F). With the expected depletion of the national standard antitoxins, replacement national standard antitoxins are produced and standardized through collaboration of the National Control Laboratory and other participants, including manufacturer(s). Therefore, Japanese National Standard Botulinum Antitoxin Type A, Equine, was replaced according to the results of a collaborative study involving the National Institute of Infectious Diseases and KM Biologics Co., Ltd. The unitage of the replacement material was determined through mouse neutralization tests, which involved toxin-antitoxin mixture injection at pH 7.0. Potency value of 440 units/vial was obtained. However, the Japanese Minimum Requirement for Biological Products was revised, and the neutralization reactions were repeated at pH 6.0, for which considerably different potency value (656 units/vial) and survival profile of mice were obtained. In September 2021, the replacement material, Japanese National Standard Botulinum Antitoxin Type A, Equine, lot 2, was established with potency value of 656 Units/vial. The impact of pH-dependent change in potency on antitoxin quality control is discussed.

Clostridial Diseases (Botulism and Tetanus).

Botulism and tetanus are the 2 primary manifestations of neurologic disease caused by clostridial toxins. Only a small dose of clostridial toxin is required to induce severe, and often fatal, disease. Consequently, definitive diagnosis of either disease is nearly impossible to achieve antemortem or postmortem; presumptive diagnosis is usually made based on physical and neurologic examination findings. Because the severity of clinical signs can worsen rapidly, prognosis worsens when therapeutic intervention is delayed. Highly effective vaccines are available against both botulism and tetanus and are critical in preventative approaches to control.

A Family Outbreak of Type E Botulism Caused by Contaminated Vacuum-Packed Ambient-Stored Chili Chicken Feet in Zhangjiakou, China.

The epidemiological investigation and laboratory-based confirmation were performed on samples from a family botulism outbreak in Zhangjiakou, Hebei province, China. Forty-four samples, including 14 samples (leftover food, and swabs taken of both food packaging bags and dishes, and serum and vomitus of the victims) related to outbreak and 30 causative food products after outbreak, were collected and analyzed. Isolation, bacterial identification, toxin detection, and whole-genome sequencing of Clostridium spp. cultured from the latter samples and animal assays were performed. Mice injected with the cultures of the leftover chili chicken feet, together with the inner layer of its packaging bag, the plate for serving it, and supernatant of two patients' serum that demonstrated the typical signs of botulism. The polyvalent anti-botulinum neurotoxin (BoNT) and the monovalent anti-BoNT/E exhibited protective effects when administered to mice. Three Clostridium botulinum cultures were obtained and verified to be positive for BoNT/E. The whole genome analysis of the isolates revealed that the classic bont/e gene orfX cluster was found to be located on the chromosomes of all three isolates. Single nucleotide polymorphism analysis suggested that these might be from the same source. Our findings indicated that this botulism outbreak occurred following the ingestion of vacuum-packed chili chicken feet contaminated with BoNT/E produced by C. botulinum.

Investigation of botulism in free-range duck farming in the Mekong Delta, Vietnam.

Background: One of the most common diseases in free-range ducks in the Mekong Delta is botulism. Botulism is a poultry disease caused by botulinum exotoxin of Clostridium botulinum. Aim: To evaluate the prevalence of botulism in free-range ducks in the Mekong Delta and the risk of infection by determining the presence of C. botulinum in the farming environment. Methods: Research was carried out on 200 duck flocks with 187,050 individuals raised freely in the fields in the provinces of the Mekong Delta, including An Giang, Can Tho, Hau Giang, and Kien Giang. The ducks were diagnosed with botulism based on clinical symptoms. To demonstrate the presence of botulinum neurotoxins and identify serotype, samples of serum and/or gut were analyzed by mouse bioassay. Samples of soil (n = 600), water (n = 600), crabs (n = 216), and snails (n = 400) were taken from the grazing regions for C. botulinum analysis by PCR assay. Results: There were 1.19% (2,235/187,050) free-range ducks in the Mekong Delta positive for botulism. Clinical symptoms of botulism including limberneck, drooping eyelids-enlarged pupils, and leg paralysis were prevalent across free-range ducks, with the frequency of 87.92% (1,965/2,235), 90.07% (2,013/2,235), and 79.78% (1,783/2,235), respectively. The lesions of pulmonary edema-hemorrhage, hemorrhagic liver, and gas-producing intestines were common, accounting for 86.19% (362/420), 95.48% (401/420), and 92.14% (387/420), respectively. Botulin toxin type C was found in a considerable number of serum samples, accounting for 40.48% (51/126). Meanwhile, the percentage of serum samples containing botulin toxin types E and D was 28.57% (36/126) and 25.40% (32/126), respectively. Clostridium botulinum was detected in the farming environment specifically 17.5% (105/600) in soil, 19.67% (118/600) in water, 8.33% (18/216) in crabs, and 3.00% (12/400) in snails. Conclusion: The free-range ducks in the Mekong Delta were at high risk of botulism because of the latent presence of C. botulinum in the farming environment.

Multi-dimensional nanoscale liquid chromatography and nano-electrospray ion-trap mass spectrometry for detection of Clostridium botulinum type C and the produced botulinum neurotoxin type C complex.

Botulinum neurotoxin types C, D and their mosaic forms C/D and D/C produced mainly by Clostridium botulinum types C and D cause botulism in animals and belong to the most toxic substances for poultry and fish. In addition to intoxications, also toxoinfections with C. botulinum types C and D play a role that should not be underestimated, especially in veterinary medicine. Contrary to other botulinum neurotoxin complexes (BT x), the biosynthesis of these types is phage-encoded. Currently, the gold standard for neurotoxin detection in cases of clinical botulism is the mouse bioassay. In the last few years, alternatives for replacing this mouse bioassay have become increasingly interesting for the detection and characterisation of botulinum neurotoxins. Therefore, immunological techniques based mainly on antibodies, PCR or mass spectral methods have been developed. In this context, the most promising development is that of different endopeptidase assays. In our study, we were able to show that the 2D-nano-LC-MS/MS method presented by Klaubert et al. 2009 especially for detecting BT x A, B, E and F in complex culture media can also be used for detecting BT x C. The focus was therefore on transferring this method to detecting BT x C and pointing out necessary modifications of this current method. For method development, we used different culture preparations and sample conditions. To find out whether BT x C is just as stable against acetic peptic pretreatment as other BT x, we used sample preparations with and without peptic pretreatment. The decisive difference to previous publications is the detection of produced BT x C directly from culture supernatant of different strains of C. botulinum type C. In addition, we present a new approach of detecting protein fragments from C3 and C2 toxin and some specific host cell proteins of the bacterium Clostridium spp. in order to specify the carrier bacterium, therefore verifying the presence of an intact neurotoxin-encoding phage also without directly detecting BT x C and thus the possibility to produce neurotoxin. Herein, we describe a new method to examine environmental samples or suspected feed samples in cases of toxoinfections as well as finding out the causes of clinical botulism. This new approach is particularly interesting for veterinary medicine, especially for diseases like chronic botulism in cows or equine grass sickness.

Confirmation of botulism diagnosis in Australian bird samples by ELISA and RT rtPCR.

We developed a sandwich ELISA that detects Clostridium botulinum C and D toxins and reverse-transcription real-time PCRs (RT-rtPCRs) that detect botulinum C and D toxin genes, respectively, to replace the mouse bioassay. The toxin genes were closely associated with the toxin molecules and used as surrogates for the presence of toxin. Samples (638) from 103 clinical cases of birds (302) with suspected botulinum toxicity came from wild birds and poultry (9 cases). Samples tested included blood serum, other body fluids, various tissues, gut contents, maggots, water, and sediment. Botulism was diagnosed in 34 cases (all of which had positive samples in the ELISA, the C toxin gene RT-rtPCR, or both assays). Botulism was suspected in 16 cases (each of which had 1 positive sample either in the ELISA or the C toxin gene RT-rtPCR). In the remaining 53 cases, no samples were positive, but botulism could not be excluded in 32 of these cases, whereas there was no indication of botulism or another diagnosis in 21 cases. The D toxin gene was not detected in any of the clinical samples. No C or D toxin genes were detected in 71 pooled cloacal swabs from 213 healthy migratory birds. The use of an ELISA that detects botulinum C and D toxins in combination with a RT-rtPCR for the botulinum C toxin gene can help confirm the diagnosis of botulism in birds.

Mortality of Western Gulls (Larus occidentalis) Associated with Botulism Type a in Coastal Southern California, USA.

A mortality event involving at least 14 Western Gulls (Larus occidentalis) was observed on 10 October 2019 on Huntington State Beach, Orange County, California, US. Clinical signs of affected gulls included generalized weakness and difficulty standing and flying. Six additional Western Gulls with similar clinical signs were admitted for rehabilitation between 24 October and 7 November, including birds from Newport Beach and Laguna Beach, south of Huntington Beach. Eleven carcasses were submitted for postmortem examination, including nine gulls collected on 10 October from Huntington Beach, one collected on 24 October from Laguna Beach, and one collected on 6 November from Newport Beach. Six of seven gulls tested were positive for Clostridium botulinum toxin type A by mouse bioassay, including five collected on 10 October from Huntington Beach and one from Laguna Beach, approximately 23 km south, on 24 October, suggesting the toxin was available to scavenging birds for nearly 2 wk following the original exposure. Botulism type C, and less commonly type E, are most frequently documented in wild birds, including waterfowl and fish-eating birds, respectively. In contrast, botulism type A is the most common cause of foodborne botulism in humans, acquired from food contaminated with C. botulinum spores, but it has not previously been associated with mortality in free-ranging wild birds.

Modification and validation of the Endopep-mass spectrometry method for botulinum neurotoxin detection in liver samples with application to samples collected during animal botulism outbreaks.

Botulinum neurotoxins (BoNTs) are the most potent toxins known and they cause the paralytic disease botulism in humans and animals. In order to diagnose botulism, active BoNT must be detected in biological material. Endopep-MS is a sensitive and selective method for serum samples, based on antibody capture, enzymatic cleavage of target peptides, and detection of cleavage products using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). In many cases of animal botulism, serum samples are not available or they do not contain detectable amounts of BoNT and liver sampling is an alternative for postmortem examinations. However, the Endopep-MS method is impaired by the inherent protease activity of liver samples. In the presented study, the Endopep-MS method has been successfully modified and validated for analysis of cattle, horse, and avian liver samples, introducing a combination of a salt washing step and a protease inhibitor cocktail. These modifications resulted in a substantial decrease in interfering signals and increase in BoNT-specific signals. This led to a substantial improvement in sensitivity for especially BoNT-C and C/D which are among the most prominent serotypes for animal botulism. Botulism was diagnosed with the new method in liver samples from dead cattle and birds from outbreaks in Sweden. Graphical Abstract.

Outbreak of botulism type A in dairy cows detected by MALDI-TOF mass spectrometry.

Twenty-eight lactating dairy cattle in New York State were exposed to botulism toxin; 12 died and 16 recovered but never returned to full productivity. Pieces of a raccoon carcass were found in the total mixed ration on the first day of the outbreak. Clinical signs included anorexia, decreased milk production, decreased tongue tone, profound weakness, and recumbency. Clostridium botulinum type A (BoNT/A) was detected in rumen contents from 2 deceased cows via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In addition, C. botulinum type C was cultured from the liver of a third cow, and C. botulinum neurotoxin-producing type C gene (bont/C) was detected via real-time PCR. On postmortem examination, 4 cows had findings suggestive of toxic myopathy, but the cause and significance of these lesions is unknown given that botulism is typically not associated with gross or histologic lesions. This outbreak of BoNT/A in cattle in North America was diagnosed via MALDI-TOF MS, a rapid and sensitive modality for detection of botulinum preformed neurotoxin.

Manure contamination with Clostridium botulinum after avian botulism outbreaks: management and potential risk of dissemination.

BACKGROUND: Persistence of Clostridium botulinum in the environment is well known. Getting rid of it after animal botulism outbreaks is so tricky, especially as far as manure concerns. This study aimed at 1. describing manure management on 10 poultry farms affected by botulism and 2. assessing the persistence of C botulinum in poultry manure after the outbreak. METHODS: Each farm was visited twice at two different manure storage times (two weeks after manure removal and two months later). Fifteen samples of manure were collected on each visit and C botulinum was detected using real-time PCR. RESULTS: Management of manure varied among poultry farms (classical storage, addition of quicklime, bacterial flora or incineration). C botulinum was detected in the manure of all 10 farms, 56.5per cent of samples being positive. C botulinum was detected significantly more frequently at the second visit (65.8per cent vs 49.7per cent, P<0.01) and on the surface of the pile (63.1per cent vs 50per cent, P=0.025). CONCLUSION: This study shows the persistence of C botulinum in poultry manure over time after a botulism outbreak and highlights manure management as a key health issue in preventing spore dissemination in the environment and recurrence of the disease.

First record of mass wild waterfowl mortality due to Clostridium botulinum in Brazilian semiarid.

In 2008, 270 wild birds from aquatic environments were found dead or debilitated on the banks of smaller lakes that had been formed due to the decrease in the level of the holding lake of the Sobradinho Dam located on the São Francisco River in the Caatinga of Bahia, Brazil. The outbreak occurred months after the dam's partial drainage, with the formation of puddles that accumulated decomposing organic material. Amongst the 270 individuals examined and/or found dead, the majority (50%) of the birds found belonged to the Anatidae family. The debilitated birds presented neurological clinical signs including lack of motor coordination, weakness, grave flaccid paralysis in the legs, wings, neck and eyelids, diarrhea, and dyspnea. Tissue samples of the birds were collected, as were water samples and samples of the substrate of the lakes. Zoonotic arboviroses or heavy metals were not detected. Analyses of liver and digestive tract content samples through bioassay and serum neutralization in mice revealed the presence of type C botulinic toxin in the viscerae samples, and type D in sediment samples. According to our knowledge, this is the first record of an outbreak of botulism in wild birds in natural conditions in Brazil.

Recombinant vaccine against botulism in buffaloes: Evaluation of the humoral immune response over 12 months.

Botulism is a neuroparalytic intoxication, usually fatal, caused by the botulinum toxins (BoNTs). Vaccination is the best-known strategy to prevent this disease in ruminants. Serotypes C and D and their variants CD and DC are the main types responsible for botulism in bovine and buffaloes in Brazil and cattle in Japan and Europe. Brazil has a herd of approximately 1.39 million buffaloes and is the largest producer in the Western world. This study aimed to assess the humoral immune response of buffaloes during the 12-month period after vaccination against BoNT serotypes C and D with a recombinant vaccine in three different concentrations (100, 200, and 400 µg) of non-purified recombinant proteins (Vrec) and also with a bivalent commercial toxoid (Vcom). Vrec400 was the best vaccine among those tested because it induced higher levels of antibodies and maintained higher levels of antibodies for the longest time, while Vrec200 could be considered the most cost-effective vaccine for large-scale production. None of the vaccines were able to promote continuous immunological protection within the timeframe proposed by the current Brazilian vaccination protocol. Further studies should focus on vaccine adjustments to ensure continued humoral protection against botulism.

Metatranscriptomics reveals that the death of a Mongolian wild ass was caused by Clostridium botulinum in Inner Mongolia, China.

Clostridium botulinum is an important pathogen that causes botulism in humans and animals worldwide. C. botulinum group III strains, which produce a single toxin of type C or D or a chimeric toxin of type C/D or D/C, are responsible for botulism in a wide range of animal species including cattle and birds. We used unbiased high-throughput RNA sequencing (i.e., metatranscriptomics) to identify a strain of group III C. botulinum from a deceased Mongolian wild ass (Equus hemionus). The strain was closely related to some European strains. Genetic analysis of the recovered bacterial sequences showed that the C. botulinum strain identified might represent a type C/D strain of group III. Infection by C. botulinum producing the mosaic toxin of type C/D is the most likely cause of the death of the wild ass.

Protective efficacy of recombinant bacterin vaccine against botulism in cattle.

Botulism is a paralytic disease caused by the intoxication of neurotoxins produced by Clostridium botulinum. Among the seven immunologically distinct serotypes of neurotoxins (BoNTs A - G), serotypes C and D, or a chimeric fusion termed C/D or D/C, are responsible for animal botulism. The most effective way to prevent botulism in cattle is through vaccination; however, the commercially available vaccines produced by detoxification of native neurotoxins are time-consuming and hazardous. To overcome these drawbacks, a non-toxic recombinant vaccine was developed as an alternative. In this study, the recombinant protein vaccine was produced using an Escherichia coli cell-based system. The formaldehyde-inactivated E. coli is able to induce 7.45 ± 1.77 and 6.6 ± 1.28 IU/mL neutralizing mean titers against BoNTs C and D in cattle, respectively, determined by mouse neutralization bioassay, and was deemed protective by the Brazilian legislation. Moreover, when the levels of anti-BoNT/C and D were compared with those achieved by the recombinant purified vaccines, no significant statistical difference was observed. Cattle vaccinated with the commercial vaccine developed 1.33 and 3.33 IU/mL neutralizing mean titers against BoNT serotypes C and D, respectively. To the best of our knowledge, this study is the first report on recombinant E. coli bacterin vaccine against botulism. The vaccine was safe and effective in generating protective antibodies and, thus, represents an industry-friendly alternative for the prevention of cattle botulism.

Development of An Innovative and Quick Method for the Isolation of Clostridium botulinum Strains Involved in Avian Botulism Outbreaks.

Avian botulism is a serious neuroparalytic disease mainly caused by a type C/D botulinum neurotoxin produced by Clostridium botulinum group III, one of the entwined bacterial species from the Clostridiumnovyisensulato genospecies. Its isolation is very challenging due to the absence of selective media and the instability of the phage carrying the gene encoding for the neurotoxin. The present study describes the development of an original method for isolating C. botulinum group III strains. Briefly, this method consists of streaking the InstaGene matrix extraction pellet on Egg Yolk Agar plates and then collecting the colonies with lipase and lecithinase activities. Using this approach, it was possible to isolate 21 C. novyi sensu lato strains from 22 enrichment broths of avian livers, including 14 toxic strains. This method was successfully used to re-isolate type C, D, C/D, and D/C strains from liver samples spiked with five spores per gram. This method is cheap, user-friendly, and reliable. It can be used to quickly isolate toxic strains involved in avian botulism with a 64% success rate and C. novyi sensu lato with a 95% rate. This opens up new perspectives for C. botulinum genomic research, which will shed light on the epidemiology of avian botulism.