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Genotyping Clostridium botulinum toxinotype A isolates from patients using amplified rDNA restriction analysis.

In this study, the application of amplified rDNA restriction analysis (ARDRA) for characterizing Clostridium botulinum toxinotype A strains isolated from individuals with botulism was evaluated. Ten restriction enzymes were tested for their suitability in ARDRA as a typing method and HhaI was selected for the best outcome. Analysis of HhaI restriction profiles of the amplified products divided C. botulinum isolates into three clusters. Non-toxigenic Clostridium sporogenes strains showed an ARDRA restriction pattern that was distinct from those observed for C. botulinum. The successful use of ARDRA for subdivision of C. botulinum in this study confirmed that this technique is a powerful method for typing of C. botulinum toxinotype A clonal diversity. In addition, it is rapid, sensitive and simple.

Bacterial Typing Techniques↗

Polymerase chain reaction for the rapid identification of Clostridium botulinum type A strains and detection in food samples.

A polymerase chain reaction (PCR) was developed for the detection of Clostridium botulinum type A, a cause of human botulism. A two primer set and an oligonucleotide detection probe were used to specifically detect Cl. botulinum type A neurotoxin gene (BoNT/A). After 40 cycles of amplification, detection of a 798 bp amplified DNA fragment was carried out by agarose gel electrophoresis and Southern blot hybridization. This assay was able to detect 12.5 fg of purified target DNA or five bacteria per reaction. The sensitivity in artificially contaminated food samples after an 18 h enrichment step ranges from 10 to 10(3) bacteria per g according to the type of food samples. No cross-reactions were observed with the other Cl. botulinum toxinotypes and other bacteria found routinely in food. This PCR method may provide a suitable and rapid alternative to standard techniques for detection of Cl. botulinum type A in food samples.

Animals↗

Clostridium botulinum and sudden infant death syndrome: a 10 year prospective study.

It has been proposed that sudden and unexpected death in infants due to intestinal infection with Clostridium botulinum may mimic the clinicopathological features of sudden infant death syndrome. Between 3.3 and 3.8% of infants in some series have had this neurotoxin-producing bacterium isolated on faecal culture. Prospective screening of 248 infants presenting with the sudden infant death syndrome to the Adelaide Children's Hospital over a 10 year period from 1981 to 1990 was conducted. Faecal samples were obtained from both small and large intestines and cultured specifically for C. botulinum. No samples were positive. The results of this study suggest that routine post-mortem culture of faeces for C. botulinum has been of limited use within the South Australian infant population over the last decade, and that occult botulism has not been a significant factor in the causation of sudden death.

Botulism↗

Comparative immunogenicity of two bivalent botulinum vaccines.

OBJECTIVE: To compare the ability of a new single-dose botulinum vaccine containing a non-mineral oil adjuvant with a single dose of a conventional botulinum vaccine product to produce antibody to Clostridium botulinum types C and D in cattle in Northern Australia. DESIGN AND PROCEDURE: One hundred and fifty Brahman steer weaners were randomly divided into two groups receiving either a single dose of CSL Bivalent Botulinum vaccine or Websters Singvac. Blood samples were collected at 0, 8 and 24 weeks and tested by antibody ELISA. The final samples were also tested by the toxin neutralisation test, to test titres of neutralising antibody. RESULTS: Six months after inoculation, cattle vaccinated with Websters Singvac had ELISA antibody response twice that of CSL conventional product. However, this difference was only evident for neutralising antibody to type C botulinum toxin. Both products produced similar titres of type D neutralising antibody after a single dose. CONCLUSION: Websters' Singvac produces a greater neutralising antibody response to type C botulism upon single inoculation than a conventional vaccine. The product produces an equivalent neutralising antibody response to type D.

Animals↗

Biodiversity of Clostridium botulinum type E strains isolated from fish and fishery products.

The genetic biodiversity of Clostridium botulinum type E strains was studied by pulsed-field gel electrophoresis (PFGE) with two macrorestriction enzymes (SmaI-XmaI and XhoI) and by randomly amplified polymorphic DNA (RAPD) analysis with two primers (OPJ 6 and OPJ 13) to characterize 67 Finnish isolates from fresh fish and fishery products, 15 German isolates from farmed fish, and 10 isolates of North American or North Atlantic origin derived mainly from different types of seafood. The effects of fish species, processing, and geographical origin on the epidemiology of the isolates were evaluated. Cluster analysis based on macrorestriction profiles was performed to study the genetic relationships of the isolates. PFGE and RAPD analyses were combined and resulted in the identification of 62 different subtypes among the 92 type E isolates analyzed. High genetic biodiversity among the isolates was observed regardless of their source. Finnish and North American or North Atlantic isolates did not form distinctly discernible clusters, in contrast with the genetically homogeneous group of German isolates. On the other hand, indistinguishable or closely related genetic profiles among epidemiologically unrelated samples were detected. It was concluded that the high genetic variation was probably a result of a lack of strong selection factors that would influence the evolution of type E. The wide genetic biodiversity observed among type E isolates indicates the value of DNA-based typing methods as a tool in contamination studies in the food industry and in investigations of botulism outbreaks.

Animals↗

Use of immunofluorescence and animal tests to detect growth and toxin production by Clostridum botulinum type E in food.

The appearance of Clostridium botulinum type E organisms and of toxin in experimentally inoculated packages of turkey roll was followed to study the time relationship between the presence of vegetative cells and the demonstration of toxin. The presence of vegetative cells was determined by immunofluorescence, and animal tests were used to assay toxin production. Growth initiated from detoxified spores of C. botulinum type E resulted in toxin formation within 24 hr. Presence of fluorescing vegetative cells and of toxin coincided from 1 to 14 days of incubation. Beginning with the next testing date, day 21, differences were observed. Toxin could be detected for a longer time than vegetative cells. Neither toxin nor organisms could be found after 56 days of incubation. The mouse lethal dose tests (MLD per gram of turkey roll) showed fluctuations in the amount of toxin present throughout the period of testing. Maximal amounts of toxin were present during the period when fluorescing organisms were also more numerous. The applications of immunofluorescence in the study and in the diagnosis of botulism is discussed.

Animals↗

Clostridium botulinum type F: isolation from venison jerky.

A Clostridium botulinum type F was isolated from the venison jerky responsible for the only type F botulism outbreak reported in the United States. The isolate differed from the prototype Langeland type F strain in being nonproteolytic.

Anaerobiosis↗

Detection of Clostridium botulinum toxin by local paralysis elicited with intramuscular challenge.

Clostridium botulinum toxin can be identified by a characteristic, acute local paralysis that follows its injection into the gastrocnemius ("calf" muscle) of mice. The local botulism can be elicited with slightly less than one-tenth the toxin amount that is needed to kill mice by the intraperitoneal (i.p.) challenge route. The practical sensitivities of the intramuscular (i.m.) versus i.p. tests are about equal, however, because maximum sample volume injectable i.m. is 0.1 ml as compared to the 0.5-ml range that can be given i.p. i.m. injection of 10 or more mouse i.p. mean lethal doses causes paralysis in about 1 h, and an i.m. injection of about 0.5 i.p. mean lethal doses causes paralysis in 3 to 4 h. Toxin neutralization by homologous type of antitoxin only can be demonstrated with an incubated mixture of toxin and antitoxin. Although not as convenient as the i.p. method for routine use to detect botulinum toxin, the i.m. method has characteristics which could make it a useful supplement to the presently accepted i.p. procedure.

Animals↗

Intraintestinal toxin in infant mice challenged intragastrically with Clostridium botulinum spores.

Conventionally raised suckling mice were injected intragastrically with 10(5) spores of a Clostridium botulinum type A culture. Botulism was not observed, but 80% or more of mice challenged when 8 to 11 days old had botulinum toxin in the large intestine 3 days later. Mice younger than 7 days or older than 15 days were resistant to the challenge. When in vivo toxin production was started by spores given to 9-day-old mice, toxin was present in the intestine at 1 through 7 days postchallenge but with greatest consistency between 1 and 4 days. Total toxin in an intestine ranged up to 1,920 50% lethal doses as titrated intraperitoneally in adult mice. The dose infecting 50% of a group of 9-day-old mice was 700 (95% confidence limits of 170 to 3,000) spores per animal. Toxin was formed in the lumen of the large intestine; it was not associated with the ileum. Injection of 10(5) spores intraperitoneally into 9-day-old mice resulted in toxin production in the large intestines of 30% of the test animals.

Animals↗

Susceptibility to enteric botulinum colonization of antibiotic-treated adult mice.

The relationship between the indigenous intestinal microflora of adults and their resistance to the enteric botulinum infection of infant botulism was studied. Orogastric challenges of 10(5) type A Clostridium botulinum spores were given to adult mice whose gut flora had been altered by feedings of a mixture of erythromycin and kanamycin sulfate. From 80 to 100% of mice became infected when challenged 15 to 60 h after antibiotic administration. The mean infective dose of 2 X 10(4) spores per mouse for challenges given 23 h after antibiotic administration contrasted with the failure of 10(6) spores to infect control mice. Botulinum-colonized mice remained asymptomatic, although colonization lasted up to 5 days, and total botulinum toxin in the gut on days 3 and 4 postchallenge averaged 3,400 and 2,200 mouse intraperitoneal mean lethal doses. The mean infective dose for inocula placed in the colon of antibiotic-treated mice was 10(3) spores per mouse, and C. botulinum multiplied in the cecum as well as in the colon.

Bacterial Physiological Phenomena↗

Significance of 12S toxin of Clostridium botulinum type E.

The pathogenesis of type E botulism is discussed as an aspect of the physicochemical and biological properties of 12S toxins (prototoxin and trypsin-activated 12S toxin) and the Ealpha and Ebeta components of each 12S toxin. A molecular weight of 350,000 was determined for each 12S toxin and 150,000 for Ealpha and Ebeta. Owing to the structure comprising the subunits Ealpha and Ebeta, 12S toxins are much more stable than Ealpha at low pH values and high temperatures. Such was also the case with type A 19S toxin and its alpha component. The Ealpha component alone accounts for the total toxicity of type E toxin. The toxic substance detected in the blood of the animals administered 12S toxins orally or parenterally was identified as Ealpha from the molecular size and the chromatographic pattern. Prototoxin escaping from detoxification in the stomach owing to the subunit structure may undergo dissociation in the intestine to release the Ealpha component. After absorption, the activated Ealpha appeared in the circulating blood without any further signs of dissociation or enzymatic digestion.

Animals↗

Isolation and enumeration of Clostridium botulinum by direct inoculation of infant fecal specimens on egg yolk agar and Clostridium botulinum isolation media.

Direct plating of stool specimens on selective (Clostridium botulinum isolation) and nonselective (egg yolk agar) media was evaluated as an aid in confirming infant botulism. C. botulinum was isolated from 13 of 14 culture-positive specimens with C. botulinum isolation and from 8 of 14 egg yolk agar. No lipase reaction was seen on plates of 31 culture-negative specimens.

Agar↗

PCR for detection of Clostridium botulinum type C in avian and environmental samples.

A PCR was developed and applied for the detection of Clostridium botulinum type C in 18 avian and environmental samples collected during an outbreak of avian botulism, and the results were compared with those obtained by conventional methodologies based on the mouse bioassay. PCR and mouse bioassay results compared well (100%) after the enrichment of samples, but PCR results directly indicated the presence of this microorganism in six samples, while only one of these contained the type C botulinal neurotoxin before enrichment. The PCR assay was sensitive (limit of detection between 15 and 15 x 10(3) spores per PCR), specific (no amplification products were obtained with other clostridia), and rapid, since sonicated and heated samples provided enough template for amplification without any DNA purification. Eleven isolates of C. botulinum type C were recovered from mallards (Anas platyrhynchos), grey herons (Ardea cinerea), and mud during investigation of this outbreak.

Animals↗

Isolation of Clostridium botulinum from Honey.

Methods for the isolation of Clostridium botulinum from honey samples are described. A total of 9 of 90 honey samples were positive for C. botulinum; 6 of the positive samples had been fed to babies who developed infant botulism.

Bacteriological Techniques↗

Surveillance of suspect animal toxicoses with potential food safety implications in England and Wales between 1990 and 2002.

The potential chemical contamination incidents investigated by the Veterinary Laboratories Agency (VLA) between 1990 and 2002 are reviewed. Incidents were identified in the course of the VLA's surveillance of food animal disease and further investigations were carried out on behalf of the Food Standards Agency in order to identify and control the contamination of food animals and animal products with chemical hazards. In total 876 incidents were investigated, of which 588 involved the poisoning of cattle with heavy metals. There were 63 incidents involving the exposure of cattle, sheep, pigs and poultry to the accidental misuse of veterinary medicines, pesticides or rodenticides, and 50 incidents involving their exposure to microbiological toxins, particularly botulism.

Animals↗

Bioterriorism: from threat to reality.

The fears and predictions of attacks with biological weapons, which were increasing at the close of the twentieth century, were transformed into reality not long after September 11, 2001, when several anthrax-laden letters were sent through the U.S. postal system. The attack challenged our medical preparedness and scientific understanding of the epidemiology of biothreat agents. It is fortunate that this was not a massive aerosol release that could have exposed hundreds of thousands. Rapid diagnoses and medical treatments limited casualties and increased survival rates, but tragically some individuals died of inhalational anthrax. Even as physicians tested new treatment regimes and scientists employed new ways of detecting anthrax and decontaminating the mail, new predictions were made for potentially even more devastating attacks with anthrax, smallpox, plague, tularemia, botulism, or hemorrhagic fever viruses. Fear gripped the nation. Law enforcement sought to find the villain(s) who sent the anthrax letters and to deter future bioterrorist attacks. The biomedical community began to seek new ways of protecting against such future threats of bioterrorism.

Animals↗

4-aminopyridine-a review.

4-aminopyridine is the first of the aminopyridines to be used in clinical practice. It blocks potassium channels and thereby increases acetylcholine, and possibly noradrenaline, release at nerve terminals. In man the drug has a significant action at the neuromuscular junction, but has little effect on the autonomic nervous system or muscle (smooth, skeletal, or cardiac) although such actions have been demonstrated in animals. It may be clinically useful in the reversal of nondepolariser blockade and a role in antibiotic associated block has been proposed. It may be used effectively as an analeptic agent. It appears to be a useful therapeutic agent in myasthenia gravis and Eaton Lambert syndrome, although of limited use in botulism. It effects on the central nervous system are considerable. These account for the major side effects of the drug which include tremor, excitability and convulsions.

4-Aminopyridine↗