Diagnostics of Clostridium botulinum (original) (raw)
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BoNT Botulinum neurotoxin BSA 2 REVIEW OF THE LITERATURE 2.1 Equine grass sickness 2.1.1 Definition Equine grass sickness or equine dysautonomia (ED) is a disease of horses, ponies and donkeys of unknown aetiology, characterized clinically by signs of autonomic dysfunction of the GI tract due to severe and extensive damage in neurons of the autonomic (mainly enteric) nervous system (Pollin and Griffiths, 1992). As its name indicates, it occurs almost exclusively in horses while at pasture and rarely reported in stabled animals (Gilmour, 1987).
Journal of Food Safety, 1991
The efficacy of an enzyme-linked immunosorbent assay ,for botulinal toxin using polyclonal antibodies was evaluated in relation to the standard mouse assay. Qualitative tests for toxin in a meat system inoculated with Clostridium botulinum and pure cultures of various aerobic and anaerobic bacterin showed that both the mouse neurotoxin and enzyme-linked immunosorbent assays detected toxin in the samples. However the ratios of mouse:ELISA activity of culture supernatant toxins qf C. botulinum showed wide disparity among strains of types A and B. Trypsin treatment resulted in a slight loss of ELISA activity but the mouse response increased. 'Mention of brand or firm names does not constitute an endorsement by the U.S. Department of Agriculture over other of a similar nature not mentioned.
Recent Developments in Botulinum Neurotoxins Detection
Microorganisms
Botulinum neurotoxins (BoNTs) are produced as protein complexes by bacteria of the genus Clostridium that are Gram-positive, anaerobic and spore forming (Clostridium botulinum, C. butyricum, C. baratii and C. argentinense spp.). BoNTs show a high immunological and genetic diversity. Therefore, fast, precise, and more reliable detection methods are still required to monitor outbreaks and ensure surveillance of botulism. The botulinum toxin field also comprises therapeutic uses, basic research studies and biodefense issues. This review presents currently available detection methods, and new methods offering the potential of enhanced precision and reproducibility. While the immunological methods offer a range of benefits, such as rapid analysis time, reproducibility and high sensitivity, their implementation is subject to the availability of suitable tools and reagents, such as specific antibodies. Currently, the mass spectrometry approach is the most sensitive in vitro method for a ra...
2018
Botulinum neurotoxins (BoNTs) are the most potent toxins known and the causative agents of the rare but potentially life-threatening disease botulism. The elaborate mode of action of BoNTs at the molecular level, their exquisite specificity for peripheral motor neurons, and their ability to effectively inhibit neurotransmitter release led to the development of BoNTs into highly valued pharmaceutical products. Both diagnostics of botulism and potency testing of pharmaceutical BoNT preparations still employ the mouse bioassay as “gold standard assay”. This animal experiment can pose a heavy burden on the animal, including a fatal outcome of testing. Additionally, several analytical disadvantages have been described. Consequently, the development of animal replacement methods is a long pursued goal which has been focused mainly on replacement methods for pharmaceutical potency testing so far. However, fundamentally different requirements and challenges apply for diagnostics of botulism...
Detection of genes encoding botulinum neurotoxins types A to E by polymerase chain reaction
Applied and Environmental Microbiology
The polymerase chain reaction (PCR) was used as the basis for the development of highly sensitive and specific diagnostic tests for organisms harboring botulinum neurotoxin type A through E genes. Synthetic DNA primers were selected from nucleic acid sequence data for Clostridium botulinum neurotoxins. Individual components of the PCR for each serotype (serotypes A through E) were adjusted for optimal amplification of the target fragment. Each PCR assay was tested with organisms expressing each of the botulinum neurotoxin types (types A through G), Clostridium tetani, genetically related nontoxigenic organisms, and unrelated strains. Each assay was specific for the intended target. The PCR reliably identified multiple strains having the same neurotoxin type. The sensitivity of the test was determined with different concentrations of genomic DNA from strains producing each toxin type. As little as 10 fg of DNA (approximately three clostridial cells) was detected. C. botulinum neurotoxin types A, B, and E, which are most commonly associated with human botulism, could be amplified from crude DNA extracts, from vegetative cells, and from spore preparations. This suggests that there is great potential for the PCR in the identification and detection of botulinum neurotoxin-producing strains.
Occurrence of Clostridium botulinum neurotoxin in chronic disease of dairy cows
Veterinary microbiology, 2015
Botulism caused by neurotoxins of Clostridium (C.) botulinum is a rare, but serious life-threatening disease in humans and animals. Botulism in livestock is usually caused by the oral uptake of C. botulinum neurotoxins (BoNT) via contaminated feed and is characterized by flaccid paralysis. In the recent past a new syndrome caused by BoNT in dairy cattle was postulated. It was supposed that C. botulinum is able to colonize the lower intestine and may subsequently produce neurotoxin. The continuous resorption of small amounts of these BoNT may then provoke the so called syndrome of "chronic" or "visceral" botulism involving unspecific clinical symptoms, reduced performance of dairy cows and massive animal losses in the affected herd. To test this hypothesis a case-control study was conducted involving 92 affected farms and 47 control farms located in Northern Germany. Fecal samples of 1388 animals were investigated for the presence of BoNT to verify the key require...
Clostridium botulinum type C, D, C/D, and D/C: An update
Frontiers in Microbiology
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, ...
International Journal of Food Microbiology, 2005
A simple, rapid, cost-effective in vitro slot blot immunoassay was developed for the detection and quantification of botulinum neurotoxin type E (BoNT/E) in cultures. Culture supernatants of 36 strains of clostridia, including 12 strains of Clostridium botulinum type E, 12 strains of other C. botulinum neurotoxin serotypes, and 12 strains of other clostridial species were tested. Samples containing BoNT/E were detected using affinity-purified polyclonal rabbit antisera prepared against BoNT/E with subsequent detection of secondary antibodies using chemiluminescence. All strains of C. botulinum type E tested positive, while all non C. botulinum type E strains tested negative. The sensitivity of the slot blot immunoassay for detection of BoNT/E was approximately four mouse lethal doses (MLD). The intensity of chemiluminescence was directly correlated with the concentration of BoNT/E up to 128 MLD, allowing quantification of BoNT/E between 4 and 128 MLD. The slot blot immunoassay was compared to the mouse bioassay for detection of BoNT/E using cultures derived from fish samples inoculated with C. botulinum type E, and cultures derived from naturally contaminated environmental samples. A total of 120 primary enrichment cultures derived from fish samples, of which 103 were inoculated with C. botulinum type E, and 17 were uninoculated controls, were assayed. Of the 103 primary enrichment cultures derived from inoculated fish samples, all were positive by mouse bioassay, while 94 were also positive by slot blot immunoassay, resulting in a 7.5% false-negative rate. All 17 primary enrichment cultures derived from the uninoculated fish samples were negative by both mouse bioassay and slot blot immunoassay. A total of twenty-six primary enrichment cultures derived from environmental samples were tested by mouse bioassay and slot blot immunoassay. Of 13 primary enrichment cultures positive by mouse bioassay, 12 were also positive by slot blot immunoassay, resulting in a 3.8% false-negative rate. All 13 primary enrichment cultures that tested negative by mouse bioassay also tested negative by slot blot immunoassay. The slot blot immunoassay could be used routinely as a