Methods for Detection of Clostridium botulinum Toxin in Foods†

2005 ◽  
Vol 68 (6) ◽  
pp. 1256-1263 ◽  
Author(s):  
SHASHI K. SHARMA ◽  
RICHARD C. WHITING
Keyword(s):  
Clostridium Botulinum ◽  
Botulinum Neurotoxin ◽  
Rapid Identification ◽  
Detection Methods ◽  
Mouse Bioassay ◽  
Large Numbers ◽  
Long Time ◽  
Botulinum Neurotoxins ◽  
Response Systems ◽  
Spore Forming Bacteria

Botulism is a deadly disease caused by ingestion of the preformed neurotoxin produced from the anaerobic spore-forming bacteria Clostridium botulinum. Botulinum neurotoxins are the most poisonous toxins known and have been a concern in the food industry for a long time. Therefore, rapid identification of botulinum neurotoxin using molecular and biochemical techniques is an essential component in the establishment of coordinated laboratory response systems and is the focus of current research and development. Because of the extreme toxicity of botulinum neurotoxin, some confirmatory testing with the mouse bioassay is still necessary, but rapid methods capable of screening large numbers of samples are also needed. This review is focused on the development of several detection methods for botulinum neurotoxins in foods.

scholarly journals  Descriptive study of botulism in an Austrian dairy herd: a case report

2012 ◽  
Vol 57 (No. 3) ◽  
pp. 143-149 ◽  
Author(s):  
J. Kummel ◽  
R. Krametter-Froetscher ◽  
G. Six ◽  
R. Brunthaler ◽  
W. Baumgartner ◽  
...  
Keyword(s):  
Veterinary Medicine ◽  
Clostridium Botulinum ◽  
Dairy Farm ◽  
Dairy Herd ◽  
Descriptive Study ◽  
Mouse Bioassay ◽  
Swallowing Difficulties ◽  
Botulinum Neurotoxins ◽  
The University ◽  
Healthy Calf

An outbreak of botulism was suspected at an Austrian dairy farm in June 2010. Six Simmental cows, out of a herd of 29, were affected and showed the typical signs. The affected cows included either animals suffering from sudden recumbency and reduced tongue tone or others which developed paresis, which gradually led to recumbency. Most of the affected animals died. Two cases were submitted to the Clinic for Ruminants at the University of Veterinary Medicine Vienna in order to clarify the cause of illness. The animals had reportedly been fed recently with silage possibly polluted by discarded cat carcasses spread onto the pasture used for the silage in question. The two referred cases both showed recumbency and swallowing difficulties. Both cows had to be euthanized. One of the two cows was in the late stage of pregnancy and a healthy calf could be delivered by Caesarean section. The outbreak of botulism was diagnosed clinically as well as by confirmation of Clostridium botulinum neurotoxins C and D by mouse bioassay. To the authors’ knowledge, this is the first report in which a live calf has been delivered out of a cow suffering from acute botulism.  

scholarly journals Immunoprecipitation of Native Botulinum Neurotoxin Complexes from Clostridium botulinum Subtype A Strains

2014 ◽  
Vol 81 (2) ◽  
pp. 481-491 ◽  
Author(s):  
Guangyun Lin ◽  
William H. Tepp ◽  
Marite Bradshaw ◽  
Chase M. Fredrick ◽  
Eric A. Johnson
Keyword(s):  
Gastrointestinal Tract ◽  
Clostridium Botulinum ◽  
Botulinum Neurotoxin ◽  
Protein Complexes ◽  
Host Strain ◽  
Mouse Serum ◽  
Content Type ◽  
Strain Nctc ◽  
Botulinum Neurotoxins ◽  
Subtype A

ABSTRACTBotulinum neurotoxins (BoNTs) naturally exist as components of protein complexes containing nontoxic proteins. The nontoxic proteins impart stability of BoNTs in the gastrointestinal tract and during purification and handling. The two primary neurotoxin complexes (TCs) are (i) TC1, consisting of BoNT, nontoxin-nonhemagglutinin (NTNH), and hemagglutinins (HAs), and (ii) TC2, consisting of BoNT and NTNH (and possibly OrfX proteins). In this study, BoNT/A subtypes A1, A2, A3, and A5 were examined for the compositions of their TCs in culture extracts using immunoprecipitation (IP). IP analyses showed that BoNT/A1 and BoNT/A5 form TC1s, while BoNT/A2 and BoNT/A3 form TC2s. AClostridium botulinumhost strain expressing recombinant BoNT/A4 (normally present as a TC2) from an extrachromosomal plasmid formed a TC1 with complexing proteins from the host strain, indicating that the HAs and NTNH encoded on the chromosome associated with the plasmid-encoded BoNT/A4. Strain NCTC 2916 (A1/silent B1), which carries both anhasilentbont/bcluster and anorfX bont/a1cluster, was also examined. IP analysis revealed that NCTC 2916 formed only a TC2 containing BoNT/A1 and its associated NTNH. No association between BoNT/A1 and the nontoxic proteins from the silentbont/bcluster was detected, although the HAs were expressed as determined by Western blotting analysis. Additionally, NTNH and HAs from the silentbont/bcluster did not form a complex in NCTC 2916. The stabilities of the two types of TC differed at various pHs and with addition of KCl and NaCl. TC1 complexes were more stable than TC2 complexes. Mouse serum stabilized TC2, while TC1 was unaffected.

scholarly journals Detection of Type A, B, E, and F Clostridium botulinum Neurotoxins in Foods by Using an Amplified Enzyme-Linked Immunosorbent Assay with Digoxigenin-Labeled Antibodies

2006 ◽  
Vol 72 (2) ◽  
pp. 1231-1238 ◽  
Author(s):  
Shashi K. Sharma ◽  
Joseph. L. Ferreira ◽  
Brian S. Eblen ◽  
Richard C. Whiting
Keyword(s):  
Immunosorbent Assay ◽  
Clostridium Botulinum ◽  
Botulinum Neurotoxin ◽  
Large Scale ◽  
Lethal Dose ◽  
Meat Products ◽  
Type A ◽  
Detection Sensitivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Botulinum Neurotoxins

ABSTRACT An amplified enzyme-linked immunosorbent assay (ELISA) for the detection of Clostridium botulinum complex neurotoxins was evaluated for its ability to detect these toxins in food. The assay was found to be suitable for detecting type A, B, E, and F botulinum neurotoxins in a variety of food matrices representing liquids, solid, and semisolid food. Specific foods included broccoli, orange juice, bottled water, cola soft drinks, vanilla extract, oregano, potato salad, apple juice, meat products, and dairy foods. The detection sensitivity of the test for these botulinum complex serotypes was found to be 60 pg/ml (1.9 50% lethal dose [LD50]) for botulinum neurotoxin type A (BoNT/A), 176 pg/ml (1.58 LD50) for BoNT/B, 163 pg/ml for BoNT/E (4.5 LD50), and 117 pg/ml for BoNT/F (less than 1 LD50) in casein buffer. The test could also readily detect 2 ng/ml of neurotoxins type A, B, E, and F in a variety of food samples. For specificity studies, the assay was also used to test a large panel of type A C. botulinum, a smaller panel of proteolytic and nonproteolytic type B, E, and F neurotoxin-producing Clostridia, and nontoxigenic organisms using an overnight incubation of toxin production medium. The assay appears to be an effective tool for large-scale screening of the food supply in the event of a botulinum neurotoxin contamination event.

scholarly journals Construction of a Nontoxigenic Clostridium botulinum Strain for Food Challenge Studies

2009 ◽  
Vol 76 (2) ◽  
pp. 387-393 ◽  
Author(s):  
Marite Bradshaw ◽  
Kristin M. Marshall ◽  
John T. Heap ◽  
William H. Tepp ◽  
Nigel P. Minton ◽  
...  
Keyword(s):  
Clostridium Botulinum ◽  
Food Industry ◽  
Botulinum Neurotoxin ◽  
Food Challenge ◽  
Laboratory Animals ◽  
Regulatory Agencies ◽  
Mouse Bioassay ◽  
Potential Threat ◽  
Western Blots

ABSTRACT Clostridium botulinum produces the most poisonous natural toxin known and is a perennial concern to the food industry and to regulatory agencies due to the potential threat of food-borne botulism. To ensure the botulinal safety of foods, rigorous food challenge testing to validate food-processing conditions and food formulations has been routinely performed. Detection of the botulinum neurotoxin is performed by using a mouse bioassay and/or in vitro assays. There has been considerable interest by the food industry and regulatory agencies in minimizing or even replacing the use of animals in these challenge studies. In addition, due to stringent select-agent regulations, the testing of various foods using toxigenic C. botulinum strains requires facilities and personnel that are certified for work with this organism. For this purpose we propose to generate sets of nontoxigenic C. botulinum strains from proteolytic and nonproteolytic groups that differ from the wild-type strains only by their inability to produce botulinum neurotoxin. In this initial study we describe the generation of a nontoxigenic mutant of C. botulinum strain 62A using the ClosTron mutagenesis system by inserting a group II intron into the botulinum neurotoxin type A gene (bont/A). The mutant clones were nontoxigenic as determined by Western blots and mouse bioassays but showed physiological characteristics, including growth properties and sporulation, that were similar to those of the parent strain in laboratory media. Additional studies will be required to evaluate comparable characteristics in various food matrices. The availability of suitable nontoxigenic C. botulinum strains for food challenge studies will be beneficial for enhancing the botulinal safety of foods as well as increasing the biosafety of workers and may eliminate the use of laboratory animals.

scholarly journals A quadruplex real-time PCR assay for rapid detection and differentiation of the Clostridium botulinum toxin genes A, B, E and F

2010 ◽  
Vol 59 (1) ◽  
pp. 55-64 ◽  
Author(s):  
Benjamin A. Satterfield ◽  
Alvin F. Stewart ◽  
Cynthia S. Lew ◽  
David O. Pickett ◽  
Marissa N. Cohen ◽  
...  
Keyword(s):  
Real Time ◽  
Clostridium Botulinum ◽  
Bacterial Species ◽  
Biological Weapons ◽  
Mouse Bioassay ◽  
Pcr Assay ◽  
Real Time Quantitative Pcr ◽  
Toxin Genes ◽  
Faecal Samples ◽  
Botulinum Neurotoxins

Clostridium botulinum is the aetiological agent of botulism, a disease marked by flaccid paralysis that can progress to asphyxiation and death. This species is defined by the production of one of the botulinum neurotoxins (BoNTs), which are the most potent toxins known. Because of their potency, these toxins have the potential to be used as biological weapons, and therefore C. botulinum has been classified as a category A select agent. There are four related but antigenically distinct BoNT types that cause disease in humans, A, B, E and F. The mouse bioassay is the current gold standard by which BoNTs are confirmed. However, this method is expensive, slow and labour-intensive. Although PCR-based assays have been used extensively for the detection of BoNT-producing bacteria in food, animals and faecal samples, and recently to help diagnose disease in humans, no real-time quantitative PCR (qPCR) assay has yet been developed that can identify and differentiate all four BoNTs that cause disease in humans. This report describes the development of a qPCR single-tube assay that uniquely identifies these four BoNTs responsible for human disease. A total of 79 C. botulinum isolates with varying toxin types was evaluated in this study, as well as numerous near-neighbours and other bacterial species. The results showed that this quadruplex assay was capable of detecting any of the four toxin genes in a given sample at a sensitivity of about 130–840 fg genomic DNA and could detect the presence of up to all four BoNT genes simultaneously in a given sample. The assay was also functional in the presence of extraneous organic matter commonly found in various environmental samples.

scholarly journals The First Case of Botulism in a Donkey

2019 ◽  
Vol 6 (2) ◽  
pp. 43
Author(s):  
Aliai Lanci ◽  
Riccardo Rinnovati ◽  
Fabrizio Anniballi ◽  
Bruna Auricchio ◽  
Concetta Scalfaro ◽  
...  
Keyword(s):  
Clostridium Botulinum ◽  
Botulinum Neurotoxin ◽  
Type B ◽  
National Reference ◽  
Reference Centre ◽  
First Case ◽  
Obligate Anaerobic ◽  
Genus Clostridium ◽  
Botulinum Type ◽  
Spore Forming Bacteria

Botulism, a severe neuroparalytic disease that can affect humans, all warm-blooded animals, and some fishes, is caused by exotoxins produced by ubiquitous, obligate anaerobic, spore-forming bacteria belonging to the genus Clostridium and named botulinum neurotoxin (BoNT)-producing clostridia. This report presents the case of a 3-year-old donkey mare referred for progressive and worsening dysphagia of four days’ duration. Her voluntary effort in eating and drinking was conserved, and she was able to slow chew without swallowing. A complete neurological examination was performed, and botulism was strongly suspected. The ability to swallow feed and water returned on the tenth day of hospitalization and improved progressively. The jenny was discharged from the hospital after fifteen days. During the hospitalization, the Italian National Reference Centre for Botulism confirmed the diagnosis: mare’s feces were positive for BoNT/B and Clostridium botulinum type B.

scholarly journals Persistence of Clostridium botulinum Neurotoxin Type E in Tissues from Selected Freshwater Fish Species: Implications to Public Health

2006 ◽  
Vol 69 (5) ◽  
pp. 1164-1167 ◽  
Author(s):  
ADAM M. YULE ◽  
JOHN W. AUSTIN ◽  
IAN K. BARKER ◽  
BRIGITTE CADIEUX ◽  
RICHARD D. MOCCIA
Keyword(s):  
Public Health ◽  
Fish Species ◽  
Clostridium Botulinum ◽  
Yellow Perch ◽  
Botulinum Neurotoxin ◽  
Round Goby ◽  
Mouse Bioassay ◽  
Commercial Fish ◽  
Rainbow Trout Oncorhynchus Mykiss ◽  
Round Gobies

Rainbow trout (Oncorhynchus mykiss), round gobies (Neogobius melanostomas), yellow walleye (Stizostedion vitreum), and yellow perch (Perca flavescens) were given Clostridium botulinum neurotoxin type E (BoNT/E) at four doses (0, 800, 1,500, and 4,000 mouse lethal doses). BoNT/E was sought in the fish tissues at death or at the conclusion of the experiment (10 days after treatment). Fish were divided into a “fillet” (axial musculature) and a “nonfillet” sample before testing for BoNT/E toxicity with a mouse bioassay. BoNT/E was detected in all species. The percentage of positive BoNT samples ranged across the species and doses from 0 (trout, perch, and walleye) to 17% (round goby) in fillet tissues and from 0 (perch) to 92% (round goby) in nonfillet tissues. The lack of positive fillet samples in three key commercial fish species suggests that the public health implications of eating these fish are minimal. However, the presence of toxin in the nonfillet compartment of a high proportion of fish supports the hypothesis that live intoxicated fish are a vehicle for the transfer of BoNT/E to fish-eating birds, which are then in turn, intoxicated.

scholarly journals Critical Analysis of Neuronal Cell and the Mouse Bioassay for Detection of Botulinum Neurotoxins

Toxins ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 713 ◽  
Author(s):  
Sabine Pellett ◽  
William H. Tepp ◽  
Eric A. Johnson
Keyword(s):  
Neuronal Cell ◽  
Basic Research ◽  
Food Samples ◽  
Detection Methods ◽  
Quantitative Detection ◽  
Mouse Bioassay ◽  
Botulinum Neurotoxins ◽  
And Performance

Botulinum Neurotoxins (BoNTs) are a large protein family that includes the most potent neurotoxins known to humankind. BoNTs delivered locally in humans at low doses are widely used pharmaceuticals. Reliable and quantitative detection of BoNTs is of paramount importance for the clinical diagnosis of botulism, basic research, drug development, potency determination, and detection in clinical, environmental, and food samples. Ideally, a definitive assay for BoNT should reflect the activity of each of the four steps in nerve intoxication. The in vivo mouse bioassay (MBA) is the ‘gold standard’ for the detection of BoNTs. The MBA is sensitive, robust, semi-quantitative, and reliable within its sensitivity limits. Potential drawbacks with the MBA include assay-to-assay potency variations, especially between laboratories, and false positives or negatives. These limitations can be largely avoided by careful planning and performance. Another detection method that has gained importance in recent years for research and potency determination of pharmaceutical BoNTs is cell-based assays, as these assays can be highly sensitive, quantitative, human-specific, and detect fully functional holotoxins at physiologically relevant concentrations. A myriad of other in vitro BoNT detection methods exist. This review focuses on critical factors and assay limitations of the mouse bioassay and cell-based assays for BoNT detection.

scholarly journals Investigation of Botulinum Neurotoxin Types from Clostridium botulinum Causing A Recent Outbreak in Vietnam

2021 ◽  
Vol 37 (4) ◽  
Author(s):  
La Thi Huong Huyen ◽  
Nguyen Thuy Linh ◽  
Nguyen Trung Thanh ◽  
Pham Yen
Keyword(s):  
Clostridium Botulinum ◽  
Stop Codon ◽  
Food Poisoning ◽  
Developed Countries ◽  
Mouse Bioassay ◽  
Gene Encoding ◽  
Pcr Products ◽  
Botulinum Neurotoxins ◽  
Specific Amplification ◽  
Foodborne Botulism

Clostridium botulinum is one of the causes of undiagnosed sudden deaths in humans due to the lethal botulinum neurotoxins (BoNTs). Foodborne botulism rarely occurs in developed countries because of being closely monitored, in opposite to developing countries including Vietnam. In the August 2020 food poisoning outbreak in Vietnam, presence of Clostridium botulinum and BoNTs was identified by culture and mouse bioassay, however, information regarding the possible toxin types was unclear. To examine the types of toxin, we designed primers for specific amplification of gene regions encoding the light chain (LC) domains for both BoNT/A and BoNT/B. After optimization, the expected PCR products were sent for sequencing. The results showed that the sequence of gene encoding BoNT/A LC was 99.2% identical to the CB-27 strain. The sequence of gene encoding BoNT/B LC was approximately 98.8% identical to reference strains. Additionally, we analyzed the sequences of the inferred proteins and identified a substitution that resulted in an early stop codon as previously found in a defective form of BoNT/B. Collectively, we provided the first evidence for C. botulinum strain possessing A(b) type in this studied outbreak. Further enzyme activity and neutralization assays are necessary to validate this preliminary toxin typing.

scholarly journals Rapid Detection of Botulinum Neurotoxins—A Review

Toxins ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 418 ◽  
Author(s):  
Robert J. Hobbs ◽  
Carol A. Thomas ◽  
Jennifer Halliwell ◽  
Christopher D. Gwenin
Keyword(s):  
Ethical Issues ◽  
Point Of Care ◽  
Control Measure ◽  
Production Quality ◽  
Detection Sensitivity ◽  
Detection Methods ◽  
Mouse Bioassay ◽  
Quality Control Measure ◽  
Botulinum Neurotoxins ◽  
The Military

A toxin is a poisonous substance produced within living cells or organisms. One of the most potent groups of toxins currently known are the Botulinum Neurotoxins (BoNTs). These are so deadly that as little as 62 ng could kill an average human; to put this into context that is approximately 200,000 × less than the weight of a grain of sand. The extreme toxicity of BoNTs leads to the need for methods of determining their concentration at very low levels of sensitivity. Currently the mouse bioassay is the most widely used detection method monitoring the activity of the toxin; however, this assay is not only lengthy, it also has both cost and ethical issues due to the use of live animals. This review focuses on detection methods both existing and emerging that remove the need for the use of animals and will look at three areas; speed of detection, sensitivity of detection and finally cost. The assays will have wide reaching interest, ranging from the pharmaceutical/clinical industry for production quality management or as a point of care sensor in suspected cases of botulism, the food industry as a quality control measure, to the military, detecting BoNT that has been potentially used as a bio warfare agent.

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