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Retrospective study of 108 cases of botulism in Poitiers, France.

Botulism, a food-borne toxin-mediated disease caused by Clostridium botulinum is still a common disease, which is most frequent in the rural environment; 108 cases, 66 males and 42 females, average age 32 years, were recorded from 1965 to 1990 in the infectious disease department of the University Hospital of Poitiers (France). In 83% of patients, the food responsible was home-cured ham. Mean incubation time was 3.4 days; digestive symptoms were observed in 93% of cases, ocular symptoms in 92% and urinary tract dysfunction in 22%. A scale of severity was used to classify the patients into those suffering from severe (6), intermediate (50) and mild (52) forms of the disease. Botulinum toxin type B was found in 36 (52%) of 69 blood samples and in 41 (51%) of 81 samples of the suspected food. From 1965 to 1976, 44 patients were treated with both toxoid and heterologous equine serotherapy. Since 1976, 29 patients have been treated with guanidine hydrochloride (35 mg/kg daily) and 35 patients with guanidine hydrochloride plus heterologous serotherapy. All 108 patients recovered without any sequelae.

Adolescent↗

Immunological characterization of the neurotoxin produced by Clostridium botulinum type A associated with infant botulism in Japan.

The neurotoxin associated with type A infant botulism in Japan shows different antigenic properties from those produced by authentic strains. The monoclonal antibodies recognizing the light chain reacted to both neurotoxins, whereas half the antibodies recognizing the heavy chain reacted specifically to the respective neurotoxin. Each neurotoxin showed its own manner of binding to brain synaptosomes. These results indicate that the distinguishable characteristics are ascribable to the heavy chain but not to the light chain. In both neurotoxins, an epitope recognized by the monoclonal antibody that reacts to the light chain and neutralizes the toxin was found to be very close to the amino-terminal half (H-1 fragment) of the heavy chain. This may support the hypothesis that the H-1 fragment functions in the transport of the light chain in the target cell.

Amino Acid Sequence↗

Application of nested polymerase chain reaction for the rapid diagnosis of infant botulism type B.

A nested polymerase chain reaction was utilized to successfully detect the type B botulinum neurotoxin gene of Clostridium botulinum in feces from a 6-month-old patient, who had already been diagnosed with type B infant botulism by mouse bio-assay. This method of rapid diagnosis without enrichment culture of feces can be applied to other types of toxins in the use of the type-specific primers. Further investigations, however, are required to define the sensitivity and specificity of the method.

Biological Assay↗

Investigation of animal botulism outbreaks by PCR and standard methods.

A double PCR procedure is proposed for identification of Clostridium botulinum C and D. This method consists of a first PCR amplification with a degenerate primer pair able to amplify a 340 bp common DNA fragment from botulinum neurotoxin (BoNT) C1 and D genes, followed by two subsequent PCR amplifications with two primer pairs specific for BoNT/C1 and D respectively (198 bp DNA fragment). This method was found to be specific for C. botulinum C and D, amongst 81 strains of C. botulinum and 21 different species of other Clostridium and bacteria tested. The detection limit ranged from 10 to 10(3) bacteria in the reaction volume according to the C. botulinum C and D strains. In 160 naturally contaminated animal and food samples submitted to a 48 h enrichment culture, the double PCR showed an 89.4% correlation rate with the standard mouse bioassay. A clear distinction between botulism type C and D was obtained. The double PCR provides a reliable alternative for detection and identification of C. botulinum C and D in clinical and food samples.

Animals↗

Single fibre EMG in 6 cases of botulism.

In 6 cases of mild botulinum intoxication, conventional EMG and single fibre EMG (SFEMG) were performed on admission to our ward (about 15 days after ingestion of the toxin) and 4, 8 and 14 weeks after admission. In 4 cases, conventional EMG resulted in abnormal findings; and they normalized 4 weeks later. On the first examination, SFEMG revealed in all cases but one the occurrence of potential pairs with abnormal jitter (above 50 mus). The % of the potential pairs with abnormal jitter ranged in different cases from 17% to 44%. Some of the potential pairs with abnormal jitter showed blockings; the occurrence of blockings was not strictly related to jitter value. Mean jitter value and % of potential pairs with abnormal jitter became progressively reduced with increasing time after intoxication. Nevertheless, in 4 cases slightly abnormal findings were still present after 4 months. The data obtained in the basal condition are in agreement with those reported by others. SFEMG findings relate fairly well to conventional EMG data and clinical status. SFEMG has proved to be a very sensitive method for studying the neuromuscular transmission defect in botulism and in obtaining further information on the course of the syndrome.

Action Potentials↗

Persistence of Clostridium botulinum type B on a cattle farm after an outbreak of botulism.

On farms involved in botulism outbreaks, cycles of Clostridium botulinum have occurred. The cycles were initiated by feeding brewers' grains contaminated with proteolytic C. botulinum type B to the cows. Spreading of manure containing feces of these cows increased the contamination of the pastures. In grass silages prepared with wilted grass from these pastures the number of C. botulinum type B organisms increased, and toxin type B was produced. Feeding cows with the contaminated silage fodder completed the cycle. Besides contamination of human foodstuffs (milk and meat), further contamination of the environment occurred. It was demonstrated that fowl may be important vectors in spreading C. botulinum.

Animal Feed↗

Evaluation of potential risk of botulism from seafood cocktails.

Clostridium botulinum E could not be detected in 35 samples of commercial seafood cocktails, ranging in pH from 4.10 to 4.85. At 30 C, toxinogenesis in homogenates acidified with a citric-acetic acid mixture was prevented at pH 4.86 or lower for crabmeat and at 5.03 or lower for shrimp. Measurements of the rate of acid penetration into the centers of large pieces of flesh indicated that the already small risk of botulism from seafood cocktails could be completely eliminated by using a cocktail sauce at a maximum pH of 3.70 and by cooling the final product to at least 10 C for 24 h.

Animals↗

Quantitative analysis of levels of serum immunoglobulin G against botulinum neurotoxin type D and association with protection in natural outbreaks of cattle botulism.

The recent outbreaks of cattle botulism in vaccinated Israeli dairy cattle prompted us to determine vaccine efficacy and reasons for vaccine failure. Analysis of clinical signs, feeding practice, vaccination history, and epidemic curves enabled us to define a study population in two outbreaks, where high doses of Clostridium botulinum neurotoxin type D (BoNT/D) were evenly consumed by the affected animal groups. Attack rates among unvaccinated 6- to 24-month-old heifers were 96% (55/57) and 85% (53/62). The attack rates in vaccinated parity 1, 2, and >or=3 cows were 40.4% (21/52), 14.3% (4/28), and 5.6% (3/54), respectively. Vaccine efficacies for these cow groups were 52.5%, 83.2%, and 93.4%, respectively. In younger, unvaccinated 2- to 6-month-old calves, presumably protected by maternal antibodies, the attack rate was 24% (17/71). These differences correlated with significant differences in levels of specific anti-BoNT/D antibody in serum by an enzyme-linked immunosorbent assay (ELISA). The ELISA performance for predicting protection was analyzed by receiver operating characteristic analysis and was found to be highly significant, with an area under the curve of 0.941 (standard error, 0.034; 95% confidence interval, 0.875 to 1.008; P < 0.000). No animals with serum ELISA unit levels above 0.33 were affected in these exposed groups. At this cutoff level, the specificity of the ELISA was 100%, sensitivity was 67%, and accuracy was 92%. We concluded that botulinum toxoids can confer adequate protection against natural exposure to lethal doses of BoNT/D; however, the vaccination protocols should be optimized. Our in-house ELISA system will enable us to optimize vaccination protocols in the animal population.

Animals↗

Microbial ecological basis of infant botulism as studied with germfree mice.

The possible role of the indigenous intestinal microflora in the toxicoinfection of human infant botulism was studied with adult germfree mice. Intraintestinal botulinum monoassociation was consistently produced when mice were fed 10 C. botulinum type A spores. Control germfree mice became enterically infected when placed in the same isolator with, but separated from, animals that had been fed spores. When transferred into a room holding a colony of normal mice, the highly susceptible gnotobiotes became resistant to challenge of 10(5) spores after about 3 days of the conventionalizing exposure. The findings are interpreted as evidence that enteric botulinum infection occurs in human infants whose intestinal tract has not yet been colonized by bacteria which are indigenous to adults and prevent growth of C. botulinum. Intestinal monoassociation could not be developed in germfree infant mice younger than 7 days.

Age Factors↗

Botulism in metronidazole- treated conventional adult mice challenged orogastrically with spores of Clostridium botulinum type A or B.

Conventional adult mice were pretreated with metronidazole to make their intestinal tracts receptive to colonization by Clostridium botulinum. These mice, in groups of 10, were fed 0 (controls), 10(2), 10(3), 10(4), or 10(5) C. botulinum type B spores and were placed for observation in filter-lid cages whose screen floors minimized the amounts of feces available for coprophagy. The opportunity to eat feces was made equal for all mouse groups by placing one mouse of every group in each of 10 cages. Mice given a spore inoculum began to develop botulism after incubation periods of slightly less than 2.75 days. Morbidity rates, which reached maxima within 5 days of challenge, were related to inocula levels. Mortality rates were also dose related. Mice given 10(5) spores and then type B antitoxin intraperitoneally, a treatment not affecting intraintestinal toxin production, remained healthy. Morbidity among control mice was seldom more than 10% and could be ascribed to toxin ingested with feces. A C. botulinum type A spore suspension gave similar results, although morbidity and mortality rates were generally lower than after challenge with a comparable number of type B spores. Mice challenged with 10(2) or 10(5) spores had similar toxin levels in their large intestines 48 h later. Morbidity rates correlated better with toxin levels in the small intestines.

Animals↗

A preclinical evaluation of aminopyridines as putative therapeutic agents in the treatment of botulism.

4-Aminopyridine and 3,4-diaminopyridine were evaluated for their abilities to delay the onset of paralysis due to botulinum neurotoxin types A, B, and E. Experiments were done on phrenic nerve-hemidiaphragm preparations excised from mice. At a concentration that produced an enhancement in muscle twitch amplitude, 4-aminopyridine and 3,4-diaminopyridine delayed the onset of paralysis due to botulinum toxin type A. Under the same conditions, the drugs did little to protect tissues against botulinum toxin types B and E. 3,4-Diaminopyridine was also evaluated for its ability to reverse the paralysis due to botulinum toxin. Experiments were done on rat phrenic nerve-hemidiaphragm preparations that had previously been poisoned in vivo. The drug produced transient increases in neuromuscular transmission, with the effect being greater for botulinum neurotoxin type A than for botulinum neurotoxin types B and E. Equivalent types of experiments were done with tetanus toxin. The results with 3,4-diaminopyridine showed that tetanus toxin resembled botulinum toxin types B and E. The data help to clarify the role of aminopyridines as therapeutic agents in the treatment of botulism. They also provide insights into the mechanism of action of clostridial neurotoxins.

4-Aminopyridine↗

Atypical toxin variant of Clostridium botulinum type B associated with infant botulism.

An atypical toxin variant of Clostridium botulinum (strain 657) was isolated from the feces of a 6-week-old female infant whose symptoms and clinical history were consistent with infant botulism. Toxin detected in the feces and the toxin produced by isolates from the feces and from two rectal swabs could be neutralized by type B botulinal antitoxin only at very high ratios of of antitoxin to toxin in the neutralization mixture. One international unit of type B antitoxin neutralized only about 10 lethal doses of 657 toxin as compared with approximately 10,000 lethal doses of conventional type B toxin from the Beans strain. Antitoxin prepared against 657 toxin was 10 times more effective against the conventional toxin than against the homologous toxin. Toxoid-antitoxin-binding studies indicate that both 657 toxin and type B toxin are heterogeneous and that both toxins may contain the same molecular variants, but that the proportions of the variants are different in each.

Antigens, Bacterial↗

Serum antibody response to Clostridium botulinum toxin in infant botulism.

A serum antibody response has not been previously demonstrated after infection with Clostridium botulinum. We developed an enzyme immunoassay for measuring serum antibody to C. botulinum toxins A, B, and E. This assay system detected a specific immunoglobulin G and immunoglobulin M antibody response to C. botulinum toxin in two patients with infant botulism.

Antibody Formation↗

Quantities of Clostridium botulinum organisms and toxin in feces and presence of Clostridium botulinum toxin in the serum of an infant with botulism.

A 7-week-old boy presented with symptoms and signs characteristic of infant botulism, and the diagnosis was confirmed by the detection of Clostridium botulinum type A organisms and toxin in the feces. The levels of organisms and toxin in the feces were measured throughout the 81-day period in hospital. The maximum levels detected were 2.46 x 10(8) C. botulinum type A colony-forming units and 64,000 mouse 100% lethal doses of type A toxin per g (wet weight) of feces. C. botulinum toxin was also detected in two samples of the patient's serum, collected 3 and 10 days after admission. Improvement in the patient's clinical condition occurred before the levels of organisms and toxin in the feces reached their maxima. A slight improvement may also have occurred while toxin was still present in the serum.

Botulinum Toxins↗

Enzyme-linked immunosorbent assay for detection of Clostridium botulinum type A and type B toxins in stool samples of infants with botulism.

An enzyme-linked immunosorbent assay (ELISA) for Clostridium botulinum type A and type B toxins was assessed for diagnostic accuracy in cases of infant botulism. This test was positive in all 22 cases confirmed by the conventional tests, which included the mouse lethality assay and stool culture. Stool specimens from five cases were positive by culture, but the mouse lethality bioassay was either negative or toxicity was judged nonspecific since it could not be neutralized by specific antitoxin. The positive ELISA results in these specimens suggested that this assay may be more reliable, in some cases, than the mouse bioassay. Of the 21 fecal specimens from suspected foodborne cases, 2 contained botulinal toxin demonstrable by the mouse assay and the ELISA. With regard to specificity, 35 fecal specimens from infants and 19 from suspected foodborne cases which were negative in the bioassay for botulinal toxins A and B were also negative in the ELISA. Only two fecal specimens with negative bioassay gave positive ELISA readings, providing a specificity rate of 96%. These results suggest that the ELISA may serve as a useful screening test to detect C. botulinum toxin in clinical specimens.

Adolescent↗

Evaluation of fluorescent-antibody tests as a means of confirming infant botulism.

Fluorescent-antibody techniques were evaluated for confirming infant botulism. Seventy-seven stool specimens from suspected cases were examined. All 34 specimens containing viable Clostridium botulinum at time of study gave positive results (29 on direct smears and 34 on enrichments). Two false-positive reactions were observed.

Botulism↗

Laboratory findings in four cases of adult botulism suggest colonization of the intestinal tract.

There was laboratory evidence of intestinal colonization in four cases of adult botulism confirmed by the Centers for Disease Control. No performed toxin was detected in available foods, but Clostridium botulinum was isolated from foods in two instances. Botulinal toxin was detected in the sera of all four patients, in one case at 47 days after ingestion of suspected food. C. botulinum was demonstrated in the stool of all four patients and persisted for 119 days after the onset of illness in one patient. Two patients had surgical alterations of the gastrointestinal tract, which may have promoted the colonization. The apparent lack of ingestion of performed toxin in these cases and the persistence of botulinal toxin or C. botulinum, or both, for long periods in three of the patients suggest that colonization of the intestinal tract occurred.

Adult↗