Table of Contents:

• Taxonomy Information
• Lifecycle Information
• Genome Information
• Biosafety Information
• Culturing Information
• Epidemiology Information
• Diagnostic Tests Information
• Infected Hosts Information
• Phinet: Pathogen-Host Interaction Network
• Lab Animal Pathobiology & Management
• References
• Data Provenance and Curators

1. Species:
   1. Clostridium botulinum (Website 1):
      1. GenBank Taxonomy No.: 1491
      2. Description: The threat posed by botulism, classically a food- and waterborne disease with a high morbidity and mortality, has increased exponentially in an age of bioterrorism. Because botulinum neurotoxin (BoNT) could be easily disseminated by terrorists using an aerosol or could be used to contaminate the food or water supply, the Centers for Disease Control and Prevention and the National Institute of Allergy and Infectious Diseases has classified it as a category A agent(Kobayashi et al., 2005). Food-borne botulism probably has accompanied mankind since its beginning. However, we have only few historical sources and documents on food poisoning before the 19th century. Some ancient dietary laws and taboos may reflect some knowledge about the life-threatening consumption of poisoned food. One example of such a dietary taboo is the 10th century edict of Emperor Leo VI of Byzantium in which manufacturing of blood sausages was forbidden. Some ancient case reports on intoxications with Atropa belladonna probably described patients with food-borne botulism, because the combination of dilated pupils and fatal muscle paralysis cannot be attributed to an atropine intoxication. At the end of the 18th century, some well-documented outbreaks of "sausage poisoning" in Southern Germany, especially in Wurttemberg, prompted early systematic botulinum toxin research. The German poet and district medical officer Justinus Kerner (1786-1862) published the first accurate and complete descriptions of the symptoms of food-borne botulism between 1817 and 1822. Kerner did not succeed in defining the suspected "biological poison" which he called "sausage poison" or "fatty poison." However, he developed the idea of a possible therapeutic use of the toxin. Eighty years after Kerner's work, in 1895, a botulism outbreak after a funeral dinner with smoked ham in the small Belgian village of Ellezelles led to the discovery of the pathogen Clostridium botulinum by Emile Pierre van Ermengem, Professor of bacteriology at the University of Ghent. The bacterium was so called because of its pathological association with the sausages (Latin word for sausage - "botulus") and not-as it was suggested-because of its shape. Modern botulinum toxin treatment was pioneered by Alan B. Scott and Edward J. Schantz(Erbguth, 2004). Over 100 years ago, the classic food-borne type was found to be caused by ingesting contaminated food containing the toxin produced by a bacteria. In the first half of the 20th century a second form, wound botulism, was discovered. Three additional forms (infant, hidden, and inadvertent) were first described in the last quarter of the 20th century(Cherington, 2004). Botulism is today divided into 5 clinical forms: classic or food-borne botulism, infant botulism, wound botulism, hidden botulism, and inadvertent botulism(Caya et al., 2004). BoNTs are serologically differentiated according to their neutralizationwith type-specific antitoxins into seven serotypes, designated by the letters A through G(Dineen et al., 2003). Based on the toxin type produced, C. botulinum strains are divided in groups I to IV, with groups I and II being the main human pathogens. Group I consists of proteolytic types A, B, and F, and group II consists of nonproteolytic types B, E, and F. The two groups are completely different in their phenotypical characteristics, such as temperature requirements, biochemical profile, and production of metabolites(Lindstrom et al., 2001). Types C and D provoke botulism in animal species, including the avian form(Arimitsu et al., 2004). All eight neurotoxins (BoNT/A to BoNT/G and TeTx (tetanus toxin)) are synthesized as a single-chain, 150,000 Da molecule which subsequently becomes nicked to the more potent dichain form, composed of a heavy (H) (approximately 100,000 Da) chain and a light (L) (50,000 Da) chain polypeptide linked by at least one disulfide bridge(Whelan et al., 1992 (a)).
      3. Variant(s):
         • Clostridium botulinum A :
           • GenBank Taxonomy No.: 36826
           • Description: Types A, B, and F are coded for by genes located on clostridial chromosomal material(Caya et al., 2004). The complete sequence of botulinum neurotoxin type A (BoNT/A; 1,296 amino acid residues, Mr 149,425). A comparison of the protein sequence revealed an overall identity of 33.8% to that of tetanus toxin (TeTx). No significant similarity to other known proteins including ADP- ribosylating toxins could be detected(Binz et al., 1990 (b)). Conservation of Cys residues flanking the position at which the toxins are cleaved to yield the heavy chain and light chain allowed the tentative identification of those residues which probably form the disulfide bridges linking the two toxin subfragments(Thompson et al., 1990). From 1990 to 2000, 160 foodborne botulism events affected 263 people in the United States, an annual incidence of 0.1 per million. Toxin type A caused 50% of all cases. The implicated foods in 40 (50%) were home-canned products (27 vegetable items, two home-canned soups, two home-canned tuna items, one each home-canned tomato juice, garlic-in-oil, and stew). Other implicated home-prepared foods included five events from potato salad or potatoes, four from homemade soup, two from homemade sausage, and one from each of the following: roast beef, liver pate, bread pudding, salsa, apple pie, hamburger, and chili. In 18 (23%) events caused by botulinum toxin type A, the specific food vehicle was not identified(Sobel et al., 2004).
         • Clostridium botulinum B :
           • GenBank Taxonomy No.: 36827
           • Description: Types A, B, and F are coded for by genes located on clostridial chromosomal material(Caya et al., 2004). Translation of the nucleotide sequence derived from all three clones demonstrated that BoNT/B was composed of 1,291 amino acids. Comparative alignment of its sequence with all currently characterized BoNTs (A, C, D, and E) and tetanus toxin (TeTx) showed that a wide variation in percent homology occurred dependent on which component of the dichain was compared. Thus, the L chain of BoNT/B exhibits the greatest degree of homology (50% identity) with the TeTx L chain, whereas its H chain is most homologous (48% identity) with the BoNT/A H chain. Overall, the six neurotoxins were shown to be composed of highly conserved amino acid domains interceded with amino acid tracts exhibiting little overall similarity. In total, 68 amino acids of an average of 442 are absolutely conserved between L chains and 110 of 845 amino acids are conserved between H chains. Conservation of Trp residues (one in the L chain and nine in the H chain) was particularly striking. The most divergent region corresponds to the extreme carboxy terminus of each toxin, which may reflect differences in specificity of binding to neuron acceptor sites(Whelan et al., 1992 (a)). During the period under study, 15 events were caused by type B botulinum toxin. The implicated foods in five (33%) of the type B toxin events were home-canned products (corn, eggs, green beans, olives, and an unspecified vegetable), and one event was due to each of the following: a commercially manufactured burrito, commercially caught and sold fish, pasta sauce and meatballs, salsa, turnip greens, and peyote. In four (25%) events, the food vehicle was not identified(Sobel et al., 2004).
         • Clostridium botulinum C :
           • GenBank Taxonomy No.: 36828
           • Description: The production of neurotoxins C, D, and E is coded for by genes carried by bacteriophage(Caya et al., 2004). The complete C I DNA sequence shows a single open readingframe which begins with an ATG at position 207 and encodes for 1291 amino acids (Mr 148,698 Da). Cys437 and Cys453 are probably involved in a disulfide bridge linking the light and heavy chains(Hauser et al., 1990). In Japan, some farmers have used ducks, named "Aigamo" in Japanese, which are cross strain of Japanese Mallard and Khaki Campbell, for reducing the chemicals in the rice. A few hundred ducks died of botulism in a certain area of Ishikawa prefecture. These ducks showed symptoms of leg and wing paralysis and became weak and listless. C. botulinum type C organisms were isolated from the contents of the gastric tract of the carcass and environmental materials such as soil, maggots, food, and (or) straw mats(Arimitsu et al., 2004). Botulism due to Clostridium botulinum type C causes considerable mortality in gulls in the UK, and refuse disposal sites are suspected as a major source of toxin(Ortiz and Smith, 1994).
         • Clostridium botulinum D :
           • GenBank Taxonomy No.: 36829
           • Description: The production of neurotoxins C, D, and E is coded for by genes carried by bacteriophage(Caya et al., 2004). The sequence of the BoNT/D gene contains a single open readingframe beginning at the ATG codon in position 47 and encodes polypeptide of 1276 amino acid residues (Mr 146,872 Da). Cys437 and Cys450 are probably involved in the disulfide bond between the light and the heavy chain. BoNT/D shares 49.6% amino acid sequence identity with BoNT/C (46.3% within the light chain, 67% within the N-terminal part of the heavy chain, and merely 35% within the putative fragment C)(Binz et al., 1990 (a)). Fifty-two feedlot cattle exhibited clinical signs suggestive of botulism. Clostridium botulinum type D organisms were recovered from ruminal fluid of 4 of the 5 affected animals tested and were isolated from bakery waste fed to the cattle. Clostridium botulinum type D has not been reported previously in Canadian cattle(Heider et al., 2001).
         • Clostridium botulinum E :
           • GenBank Taxonomy No.: 36830
           • Description: The production of neurotoxins C, D, and E is coded for by genes carried by bacteriophage(Caya et al., 2004). Translation of the sequence has shown botulinum neurotoxin type E (BoNT/E) to consist of 1252 amino acids and, as such, represents the smallest BoNT characterized to date. The light chain of the toxin exhibits the highest level of sequence similarity to tetanus toxin (TeTx, 40%). The light chains of BoNT/A and BoNT/D share 33% similarity with BoNT/E, while BoNT/C exhibits 32% similarity. In contrast, the TeTx heavy chain exhibits the lowest degree of similarity (35%) with BoNT/E, with the BoNT heavy chains sharing 46%, 36% and 37%, for neurotoxin types A, C and D, respectively(Whelan et al., 1992 (b)). From 1990 to 2000, 160 foodborne botulism events affected 263 people in the United States. During the period under study, 58 botulism events occurred in Alaska; 103 persons were affected, with a median of 5 events (range 015) and 8 cases (range 020) occurring per year. Forty-nine (84%) events and 91 (88%) cases were caused by toxin type E, all involving foods from aquatic animals. All identified foods were homemade Alaska Native foods. Eleven events, affecting 21 persons, were caused by foods consisting of fermented aquatic mammal tissues, such as mukluk (whale skin and blubber), beaver tail, and seal flipper. Fourteen events, affecting 20 persons, were caused by seal oil; 14 events, affecting 28 persons, were caused by fish foods such as fermented salmon heads and whitefish; 7 events, affecting 18 persons, were caused by fermented fish eggs; and 3 events, affecting 5 persons, were caused by foods with mixed ingredients(Sobel et al., 2004). Clostridium botulinum type E was found in 81% of sea and 61% of freshwater samples. No other toxinotypes were found. These findings indicate the possibility of Clostridium botulinum type E multiplication or at least, suitable conditions for spore survival, in anoxic sediments(Hielm et al., 1998).
         • Clostridium botulinum F :
           • GenBank Taxonomy No.: 36831
           • Description: Types A, B, and F are coded for by genes located on clostridial chromosomal material(Caya et al., 2004). Primers designed to conserved regions of botulinum and tetanus clostridial toxins were used to amplify DNA fragments from non-proteolytic Clostridium botulinum type F (202F) DNA using polymerase chain reaction technology. The fragments were cloned and the complete nucleotide sequence of the gene encoding type F toxin determined. Analysis of the nucleotide sequence demonstrated the presence of an open frame encoding a protein of 1274 amino acids, similar to other botulinum neurotoxins. Upstream of the toxin gene is the end of an open reading frame which encodes the C-terminus of a protein with homology to non-toxic-non-hemagglutinin component of type C progenitor toxin(East et al., 1992). Botulism caused by type F botulinum toxin accounts for less than 0.1% of all human botulism cases and is rarely reported in the literature. The treating physician initially considered the possibility of paralytic shellfish poisoning due to a report of shellfish ingestion, which was later determined to be frozen shrimp and a can of tuna, but no gastroenteritis or paresthesias were present(Richardson et al., 2004).
         • Clostridium botulinum G :
           • GenBank Taxonomy No.: 29341
           • Description: The gene responsible for type G neurotoxin is present on a plasmid(Caya et al., 2004). The neurotoxin gene from Clostridium botulinum type G was cloned as a series of overlapping DNA fragments generated using polymerase chain reaction (PCR) technology and primers designed to conserved regions of published botulinal toxin (BoNT) sequences. Translation of the nucleotide sequence derived from the cloned PCR fragments demonstrated that the gene encodes a protein of 1297 amino acid residues. Comparative alignment of the determined BoNT/G sequence with those of other clostridial neurotoxins revealed highest sequence relatedness (approx. 58% amino acid identity) with BoNT/B of proteolytic and non-proteolytic C. botulinum. Tetanus toxin and other BoNT types revealed lower levels of relatedness with BoNT/G (approximate range 35-42% amino acid identity)(Campbell et al., 1993). The last antigenic toxin type to be discovered wastype G. Clostridium botulinum producing neurotoxin type G was originally isolated from soil samples in Argentina,(Bhandari et al., 1997). now recognized as Clostridium argentinense(Dineen et al., 2003).

1. General biosafety information
   1. Applicable: Clostridium botulinum Precautions(Caya et al., 2004).
   2. Precautions: BSL-2 requirements state that all microbiologic laboratory procedures that may generate aerosols (such as blending, shaking, or vortexing) should be conducted in a class II biological safety cabinet, with a laboratory coat, disposable surgical gloves, and a face shield(Caya et al., 2004).
   3. Disposal: The botulinum neurotoxin may be inactivated by 0.2 M sodium hydroxide. Clostridium botulinum is inactivated by a 1:10 dilution of household bleach with a 20-minute contact time. For successful inactivation of both organism and toxin, both bleach and sodium hydroxide must be applied for a total of 40 minutes(Caya et al., 2004).

1. Costridium botulinum Culturing :
   1. Description: Trypticase-Peptone-Glucose-Yeast Extract Broth (TPGY).
   2. Medium: Trypticase-Peptone-Glucose-Yeast Extract Broth (TPGY).Trypticase 50 g,Peptone 5 g,Yeast extract 20 g,Dextrose 4 g,Sodium Thioglycollate 1 g,Distilled water 1 liter.Before sterilization (121C for 15 min), anaerobic conditions are created by boiling the medium for 10 min and, during cooling, flushing the medium with nitrogen gas(Dahlenborg et al., 2001).
   3. Optimal Temperature: 37C(Dahlenborg et al., 2001).

1. Outbreak Locations:
   1. From 1973 through 1996 in the United States, 724 cases of foodborne botulism (median, 24 cases annually (range, 8 to 86 cases)), 103 cases of wound botulism (median, 3 cases annually (range, 0 to 25 cases)), 1444 cases of infant botulism (median, 71 cases annually (range, 0 to 99 cases)), and 39 cases of botulism of undetermined type were reported to the Centers for Disease Control and Prevention (CDC).In the United States, approximately half of the cases of foodborne botulism are caused by toxin type A; the remaining foodborne cases are almost equally divided between toxins type E and type B. Among cases of infant botulism, approximately half are caused by toxin type A and half by toxin type B; among cases of wound botulism, approximately 80% are caused by toxin type A and 20% by toxin type B. In the United States, type A botulism is most common west of the Mississippi River, and type B is most common east of the Mississippi River. Type E outbreaks are most common in Alaska(Shapiro et al., 1998).
2. Transmission Information:
   1. From: Contaminated food(Shapiro et al., 1998). , To: Human(Shapiro et al., 1998). (Shapiro et al., 1998)
      Mechanism: Foodborne Botulism. Foodborne botulism is caused by ingestion of preformed toxin produced in food by C. botulinum. The most frequent source is home-canned foods, in which spores that survive an inadequate cooking and canning process germinate, reproduce, and produce toxin(Shapiro et al., 1998). Spores of C. botulinum are ubiquitous in the environment, but growth and elaboration of toxin occur only under particular conditions that include an anaerobic, low-salt, low-acid environment. The canning and fermentation of foods are particularly conducive to creating anaerobic conditions that allow C. botulinum spores to germinate. Foodborne botulism, while rare, remains a public health emergency because of its severity and epidemic potential. Home-canned foods and Alaska Native foods remain the leading causes in the United States, and restaurant-associated outbreaks continue to account for a disproportionate number of illnesses(Sobel et al., 2004).
   2. From: Contaminated wound. Contaminated wound(Caya et al., 2004). , To: Human(Caya et al., 2004). (Caya et al., 2004)
      Mechanism: Wound Botulism (WB). Since 1988, California has experienced a dramatic increase in wound botulism associated with injecting "black tar" heroin (BTH), a dark, tarry form of the drug. Among the 26 patients, the median age was 41.5 years, 15 (58%) were women, 14 (54%) were non-Hispanic white, 11 (42%) were Hispanic, and none were positive for the human immunodeficiency virus. Nearly all participants (96% of patients and 97% of controls) injected BTH, and the mean cumulative dose of BTH used per month was similar for patients and controls (27 g and 31 g, respectively; P~.6). Patients were more likely than controls to inject drugs subcutaneously or intramuscularly (92% vs 44%, P less .001) and used this route of drug administration more times per month (mean, 67 vs 24, P less .001), with a greater cumulative monthly dose of BTH (22.3 g vs 6.3 g, P less .001). A dose-response relationship was observed between the monthly cumulative dose of BTH injected subcutaneously or intramuscularly and the development of WB ({chi}2 for linear trend, 26.5; P less .001). In the final regression model, subcutaneous or intramuscular injection of BTH was the only behavior associated with WB among IDUs (odds ratio, 13.7; 95% confidence interval, 3.0-63.0). The risk for development of WB was not affected by cleaning the skin, cleaning injection paraphernalia, or sharing needles. From 1951 through 1995, 68 cases of WB were reported to California Department of Health Services (CDHS). An average of 0.49 WB cases per year were reported from 1951 through 1987; 2.25 cases per year were reported in 1988 through 1991, 3 cases in 1992, 4 in 1993, 11 in 1994, and 23 in 1995. 18 From 1988 through 1995, only 2 WB cases among IDUs were reported from outside California, and they occurred in Arizona (Foodborne and Diarrheal Disease Branch, Centers for Disease Control and Prevention, unpublished data, 1996)(Passaro et al., 1998). Under conditions of tissue necrosis and anaerobiosis, such as those seen in a subcutaneous abscess, C. botulinum spores can germinate and produce neurotoxin in vivo, with the same clinical features as those seen in food-borne botulism, except for a lack of acute gastrointestinal signs and symptoms(Caya et al., 2004).
   3. From: Intestinal contamination of infant. Intestinal contamination of infant(Caya et al., 2004). , To: Human(Caya et al., 2004). (Caya et al., 2004)
      Mechanism: Infant botulism, first described in 1976, is now the most frequently reported form(Cherington, 1998). Since 1980, it has become the most common form of botulism reported in the United States. Unlike food-borne botulism, the infant form is a combination infection intoxication, including ingestion of C botulinum spores, germination within the gastrointestinal tract, and in vivo production of toxin; these events occur in part due to the lack of a protective gastrointestinal bacterial flora and in part due to the relatively reduced levels of clostridial-inhibiting bile acid as compared to the adult gastrointestinal tract(Caya et al., 2004). The source of ingestion is unknown in approximately 85% of cases; in up to 15% of cases, the ingestion of honey is suspected(Shapiro et al., 1998). A significant risk factor for the development of infant botulism is honey consumption; 15% to 25% of honey products harbor botulinum spores (especially type B)(Caya et al., 2004). In addition, honey samples across the United States have tested positive for Clostridium botulinum spores and toxins. Such substantial evidence led the CDC to recommend that honey not be given to infants younger than 12 months old. It is important that clinicians be familiar with this risk and should not recommend honey-containing products or supplements or the use of honey as a flavoring agent for infants in this age group(Tanzi and Gabay, 2002).
   4. From: Intestinal contamination of adult. Intestinal contamination of adult(Caya et al., 2004). , To: Human(Caya et al., 2004). (Caya et al., 2004)
      Mechanism: Hidden Botulism. Hidden botulism (also known as intestinal colonization botulism) refers to those cases of botulism in adults for which there is no readily apparent source of botulinum toxin. Many of these patients may actually represent an adult form of infant botulism (also known as adult form of intestinal botulism). Clostridium botulinum organisms are present within the gastrointestinal tracts of these patients, where they proliferate and produce neurotoxin in vivo(Caya et al., 2004). Such patients often have a history of abdominal surgery, gastrointestinal tract abnormalities, or recent antibiotic treatment that may disrupt the natural gastrointestinal flora(Shapiro et al., 1998).
   5. From: Accidental exposure of human. Accidental exposure of human(Caya et al., 2004). , To: Human(Caya et al., 2004). (Caya et al., 2004)
      Mechanism: Inadvertent botulism. Inadvertent botulism, the most recent type to be recognized by the medical community, is either an iatrogenic disease (resulting from the therapeutic use of botulinum toxin) or occurring as an accidental exposure in laboratory workers. Indeed, the literature includes a recent report of full-blown botulism resulting from therapeutic botulinum toxin use in at least 2 patients. Three cases of botulism have been reported in laboratory workers who apparently contracted the disease by inhalation of the toxin; an inhalational route provides a potential basis for the use of botulinum toxin as an agent of bioterrorism or biocrime warfare(Caya et al., 2004).
3. Environmental Reservoir:
   1. Clostridium botulinum Environmental Reservoir:
      1. Description: These neurotoxigenic organisms are anaerobic, gram-positive, spore-forming bacilli and are commonly found in soils throughout the world(Shapiro et al., 1998). Clostridium botulinum spores are quite widespread throughout the world, inhabiting soil as well as both freshwater and saltwater mud(Caya et al., 2004). The prevalence of C. botulinum in Swedish cattle was established to be 73% for non-proteolytic type B and less than 5% for types E and F. Twenty-eight (64%) of the positive faecal samples had a spore load of less than 4 spores/g. Statistical analysis (ANOVA) showed that seasonal variation (summer and winter) had a significant effect on the prevalence of C. botulinum type B in cattle, whereas the effect of geographical location of rearing of the cattle (southern and central Sweden) was less significant(Dahlenborg et al., 2003).
      2. Survival: These very resilient spores are capable of surviving for up to 2 hours at 100C. The botulinum neurotoxin may be inactivated by 0.2 M sodium hydroxide. Clostridium botulinum is inactivated by a 1:10 dilution of household bleach with a 20-minute contact time. For successful inactivation of both organism and toxin, both bleach and sodium hydroxide must be applied for a total of 40 minutes(Caya et al., 2004).
4. Intentional Releases:
   1. Intentional Release Information:
      1. Description: Botulinum neurotoxin is considered the most potent lethal substance known. It is 15 000 to 100 000 times more toxic than sarin, the potent organophosphate nerve agent. Studies in monkeys indicate that, if aerosolized, botulinum toxin also can be absorbed through the lungs; this could occur in the case of a terrorist attack(Shapiro et al., 1998). It is sobering to consider that botulinum toxin is a potentially effective agent for the mass destruction of human life in biological warfare/bioterrorism contexts and is indeed categorized as a biothreat level A biological warfare agent.Botulinum toxin is not only highly lethal after ingestion of minute amounts (in water and/or food), but can also cause disease via the inhalational route and therefore potentially lends itself to biowarfare/bioterrorism activity. Indeed, unsuccessful attempts to utilize botulinum toxin in the bioterrorism context occurred in Japan on at least 3 occasions between 1990 and 1995. In 1995. In the same year, the Aum Shinrikyo cult in Japan prepared botulinum toxin before its attack on the Tokyo subway system, even though it instead chose to deploy the nerve agent sarin.Although bioterrorism-related botulinum toxin could be introduced into its target population by contaminating food and water supplies, these vectors are associated with significant limitations, including logistical difficulties. Therefore, most health care experts and government officials have focused their strongest bioterrorism-related concerns on the inhalational delivery of botulinum toxin into a target population.It has been suggested that inhaled, aerosolized toxin is typically not identifiable in either stool or serum(Caya et al., 2004).

1. Organism Detection Test:
   1. Light microscopy :
      1. Description: Organisms from the growth are examined by Gram stain or phase-contrast microscopy. Clostridium botulinum is a gram-positive, straight rod. Spores may be demonstrated by phase-contrast microscopy of wet mounts, where they appear mature and refractile(Caya et al., 2004).
2. Immunoassay Test:
   1. amp-ELISA :
      1. Description: An amplified enzyme-linked immunosorbent assay (amp-ELISA) was compared with the AOAC Official Method 977.26 for detection of Clostridium botulinum and its toxins in foods. Eleven laboratories participated and the results of 10 laboratories were used in the study. Two anaerobic culture media, tryptone-peptone-glucose-yeast extract (TPGY) and cooked meat medium (CMM) were used to generate toxic samples with types A, B, E, and F strains. The toxicity of each botulinal culture was determined by the AOAC method, and the cultures were then diluted, if necessary, to high (about 10,000 minimal lethal dose [MLD]/mL) and low (about 100 MLD/mL) test samples. The overall sensitivity of detection in TPGY and CMM cultures with the amp-ELISA was 94.7% at about 100 MLD/mL and 99.6% for samples with more or equal 10,000 MLD/mL toxicity. The amp-ELISA detection sensitivity for low toxin samples was 92.3% in TPGY and 99.4% in CMM. The amp-ELISA could be used to screen suspect cultures for botulinum toxin(Ferreira et al., 2003).
      2. False Positive: The false-positive rate ranged from 1.5% for type A to 28.6% for type F in TPGY, and from 2.4% for type A to 11.4% for type F in CMM. Most of the cross-reactivity was due to detection of other types, especially in high toxin samples(Ferreira et al., 2003).
      3. Antigen:
         • Neurotoxin A
           • Antigen GenBank Accession Number: AAQ16531 (Find similar sequence in our pathogen protein library)
         • Neurotoxin B
           • Antigen GenBank Accession Number: AAQ16536 (Find similar sequence in our pathogen protein library)
         • Neurotoxin E
           • Antigen GenBank Accession Number: AAQ16539 (Find similar sequence in our pathogen protein library)
         • Neurotoxin F
           • Antigen GenBank Accession Number: AAS59788 (Find similar sequence in our pathogen protein library)
   2. Immuno-PCR-ELISA :
      1. Description: Immuno-PCR, using the specificity of an antibody, and a reporter DNA molecule amplified by PCR, enabled the development of a sensitive assay to detect BTx-A antigen. This method combines the amplification power of PCR and a method, similar to enzyme-linked immunosorbent assay (ELISA), which detects an antigen-antibody reaction; however, instead of an enzyme being conjugated to an antibody, a reporter DNA was used which could be amplified by PCR. Anti-BTx-A antibody-DNA conjugates were synthesized using a heterobifunctional cross-linker reagent to covalently link the reporter DNA and the antibodies. The antibody-DNA conjugates with antigens were amplified by PCR, and dose-dependent relationships for each analyte were demonstrated. Detection limits of immuno-PCR for BTx-A (3.33 x 10(-17) mol) exceeded the conventional enzyme-linked immunosorbent assay (3.33 x 10(-14) mol) by a 1000-fold enhancement in detection sensitivity(Wu et al., 2001).
3. Nucleic Acid Detection Test:
4. Other Test:
   1. The Standard Lab Test for Botulism :
      1. Description: Clinical diagnosis of botulism is confirmed by specialized laboratory testing that often requires days to complete. At present, laboratory diagnostic testing for botulism in the United States is available only at the CDC and approximately 20 state and municipal public health laboratories. The laboratory should be consulted prospectively about specimen collection and processing. Samples used in diagnosis of botulism include serum (more or equal to 30 mL of blood in "tiger"-top or red-top tubes from adults, less from children), stool, gastric aspirate, and, if available, vomitus and suspect foods. Serum samples must be obtained before therapy with antitoxin, which nullifies the diagnostic mouse bioassay. An enema may be required to obtain an adequate fecal sample if the patient is constipated. Sterile water should be used for this procedure because saline enema solution can confound the mouse bioassay. Gastric aspirates and, perhaps, stool may be useful for detecting inhaled aerosolized botulinum toxin released in a bioterrorist attack. A list of the patient's medications should accompany the diagnostic samples because anticholinesterases, such as pyridostigmine bromide, and other medicines that are toxic to mice can be dialyzed from samples before testing. All samples should be kept refrigerated after collection.The standard laboratory diagnostic test for clinical specimens and foods is the mouse bioassay, in which type-specific antitoxin protects mice against any botulinum toxin present in the sample. The mouse bioassay can detect as little as 0.03 ng of botulinum toxin and usually yields results in 1 to 2 days (range, 6-96 hours). Fecal and gastric specimens also are cultured anaerobically, with results typically available in 7 to 10 days (range, 5-21 days). Toxin production by culture isolates is confirmed by the mouse bioassay.Foods suspected of being contaminated should be refrigerated until retrieval by public health personnel. The US Food and Drug Administration and the US Department of Agriculture can assist other public health laboratories with testing of suspect foods by using methods similar to those applied to clinical samples(Arnon et al., 2001).

1. Human
   1. Taxonomy Information:
      1. Species:
         1. Homo sapiens :
            • Common Name: Homo sapiens
            • GenBank Taxonomy No.: 9606
            • Description: It should be emphasized that botulism (in any form, including inhalational) is a noncontagious disease that cannot be transmitted from affected to nonaffected persons, and that the botulinum toxin cannot pass through intact skin(Caya et al., 2004).
   2. Infection Process:
      1. Infectious Dose: Clostridium botulinum organisms elaborate a very potent neurotoxin that is one of the most (if not the most) potent toxins known to man; as little as 10 pg is sufficient to kill a mouse, and the estimated ingested human toxic dose is 1 ng/kg body mass(Caya et al., 2004),
      2. Description: Botulism is a neuroparalytic illness caused by a neurotoxin produced from the anaerobic, spore-forming bacterium Clostridium botulinum(Shapiro et al., 1998), Growth from bacterial spores occurs through a number of steps that are distinguishable but poorly understood. The first of these steps is germination. As the safety margin of minimally heat-processed refrigerated foods is small, it is imperative that spore germination is well understood for non-proteolytic C. botulinum, the pathogen of most concern for the safety of these foods.When germination occurred, it proceeded rapidly, with little further germination after 6 h. Heat activation increased the initial germination rate, and increased or had no effect on the final extent of germination(Plowman and Peck, 2002), FOOD-BORNE BOTULISM.This form occurs after ingestion of food containing preformed neurotoxin, produced by clostridial organisms that contaminate inadequately processed food. The archetypical example of food-borne botulism is that resulting from ingestion of improperly prepared home-canned foodstuffs(Caya et al., 2004),
   3. Disease Information:
      1. Botulism :
         1. Incubation: Onset usually occurs 18 to 36 hours after exposure (range, 6 hours to 8 days)(Shapiro et al., 1998),
         2. Prognosis:
            The prognosis for botulism has improved greatly during the past 50 years, coincident with the advent of modern supportive care measures provided in the intensive care setting, especially featuring pulmonary function protection with the mechanical ventilator. Thus, the case fatality rate for food-borne botulism has decreased from the 50% to 70% range (worse for C botulinum neurotoxin type A) to the 5% to 20% range seen today. Infant botulism and wound botulism currently have case fatality rates of approximately 15% and 1%, respectively. It should be stressed that survival of an episode of botulism provides essentially no immunity and therefore offers no protection from future bouts of this disease(Caya et al., 2004),
         3. Diagnosis Summary: CLINICAL FINDINGS. Botulism is underdiagnosed because many clinicians are unfamiliar with the disease and because symptoms can be mistaken for more common clinical entities, such as stroke or the Guillain-Barre syndrome. However, the diagnosis of botulism is not difficult in most cases once it has been considered. Botulism should be suspected in a patient with acute onset of gastrointestinal, autonomic (such as dry mouth or difficulty focusing eyes), and cranial-nerve (diplopia, dysarthria, dysphagia) dysfunction. The diagnosis is even more likely if the patient has recently eaten home-canned foods or if family members or companions who have shared meals are similarly ill.ANCILLARY TESTING. The diagnosis of botulism is supported by ancillary testing, such as documentation of a normal result on magnetic resonance imaging or computed tomography of the brain to rule out stroke syndrome; a normal result on lumbar puncture to differentiate botulism from the Guillain-Barre syndrome, which typically causes elevated levels of protein in the cerebrospinal fluid (although protein levels may be normal initially); and a negative edrophonium chloride test result to rule out myasthenia gravis (although transient responses may occasionally be noted in botulism). Electromyography usually reveals decreased amplitude of action potentials in affected muscle groups, but this finding is relatively nonspecific. An incremental increase in amplitude to rapid repetitive electromyography by using frequencies of 20 to 50 Hz is more helpful and may distinguish botulism from the Guillain-Barre syndrome or myasthenia gravis but not the Eaton-Lambert syndrome. Electromyography should be performed by a person experienced in performing rapid repetitive testing.In most cases, lumbar puncture and brain imaging can be performed within hours of presentation. Negative results may raise the clinical suspicion for botulism and should prompt close monitoring for respiratory compromise; rapid repetitive electromyography; and, possibly, edrophonium chloride testing. State or local health officials should be contacted to discuss potential measures for preventing additional cases; the possible release of antitoxin by CDC; and the collection of serum and stool samples at the earliest possible opportunity to confirm the diagnosis of botulism by the detection of toxin if none of the ancillary tests is pathognomonic.TOXIN - MICROBIOLOGICAL TESTING. In cases of suspected foodborne botulism, serum and stool specimens and epidemiologically implicated foods should be tested for botulism neurotoxin. The most reliable method for the detection of toxin is the mouse inoculation test; this can be performed at the CDC or some state public health laboratories. Botulinum toxin type is determined by neutralizing the biological activity of toxic samples injected into mice with type-specific botulism anti-toxin. Symptoms of botulism and death occur in mice injected with unneutralized samples but not in mice injected with neutralized samples. Toxin is detected in serum or stool specimens in approximately 46% of clinically diagnosed cases. Stool specimens also should be cultured for C. botulinum because a positive C. botulinum culture from stool is also considered confirmatory for botulism. Isolation of neurotoxigenic organisms from stool specimens increased the sensitivity of laboratory testing to 73% in one case series and 67% in another. Detection of botulinum toxin from epidemiologically implicated food may provide additional confirmatory evidence for botulism; however, the isolation of C. botulinum organisms from a food devoid of toxin usually has little significance because spores are ubiquitous in the environment. If wound botulism is suspected, such specimens as wound exudate, a tissue sample, or a swab sample should be obtained for anaerobic culture in addition to a serum toxin assay. A stool specimen may be examined to exclude food or intestinal colonization as sources of toxin. Infant botulism should be suspected in any infant with constipation, poor feeding, diminished sucking and crying ability, neck and peripheral muscle weakness, or ventilatory distress. Stool cultures for C. botulinum and testing for the presence of toxin in stool should be performed in such patients(Shapiro et al., 1998), GENERAL PRINCEPELS. Appropriate samples must usually be sent to a reference laboratory, typically a state health department laboratory facility or the CDC. Laboratory evaluation includes anaerobic culture and toxin assays of appropriate samples. Specimens should be obtained and sent to a reference laboratory after consultation with the state epidemiologist, the state health department, and the CDC (emergency telephone number for the CDC: (770) 488-7100.SPECIMEN TYPES. Food should be submitted in its original container. If the original container is not available, the food should be put into a sterile, unbreakable, leak-proof container. In the rare event of a suspect commercial food product, the unopened food container should be sent immediately to the Food and Drug Administration.SPECIMEN TRANSPORT. Clinical specimens should be packaged in an insulated container with refrigerant and transported to the laboratory as quickly as possible. All specimens for isolation of C botulinum should be collected in an anaerobic transport medium. If there is an unavoidable delay in transport, serum or feces may be frozen and shipped on dry ice; freezing clinical specimens will not interfere with toxin detection but may compromise microbiologic culture detection of clostridia(Caya et al., 2004),
         4. Symptom Information :
            • Description: BOTULISM SYMTOMS IN ADULTS:.
            • Symptom -- Fatigue (Arnon et al., 2001):
              • Observed:
                77 %(Arnon et al., 2001),
            • Symptom -- Dizziness (Arnon et al., 2001):
              • Observed:
                51%(Arnon et al., 2001),
            • Symptom -- Double vision (Arnon et al., 2001):
              • Observed:
                91%(Arnon et al., 2001),
            • Symptom -- Blurred vision (Arnon et al., 2001):
              • Observed:
                65%(Arnon et al., 2001),
            • Symptom -- Dysphagia (Arnon et al., 2001):
              • Observed:
                96%(Arnon et al., 2001),
            • Symptom -- Dry mouth (Arnon et al., 2001):
              • Observed:
                93%(Arnon et al., 2001),
            • Symptom -- Dysarthria (Arnon et al., 2001):
              • Observed:
                84%(Arnon et al., 2001),
            • Symptom -- Sore throat (Arnon et al., 2001):
              • Observed:
                54%(Arnon et al., 2001),
            • Symptom -- Dyspnea (Arnon et al., 2001):
              • Observed:
                60%(Arnon et al., 2001),
            • Symptom -- Constipation (Arnon et al., 2001):
              • Observed:
                73%(Arnon et al., 2001),
            • Symptom -- Nausea (Arnon et al., 2001):
              • Observed:
                64%(Arnon et al., 2001),
            • Symptom -- Vomiting (Arnon et al., 2001):
              • Observed:
                59%(Arnon et al., 2001),
            • Symptom -- Abdominal cramps (Arnon et al., 2001):
              • Observed:
                42%(Arnon et al., 2001),
            • Symptom -- Diarrhea (Arnon et al., 2001):
              • Observed:
                19%(Arnon et al., 2001),
            • Symptom -- Arm weakness (Arnon et al., 2001):
              • Observed:
                73%(Arnon et al., 2001),
            • Symptom -- Leg weakness (Arnon et al., 2001):
              • Observed:
                69%(Arnon et al., 2001),
            • Symptom -- Paresthesia (Arnon et al., 2001):
              • Observed:
                14%(Arnon et al., 2001),
         5. Treatment Information:
            • Antitoxin therapy : The administration of antitoxin is the only specific pharmacologic treatment available for botulism. The currently available licensed antitoxin is an equine product with antibodies to toxin types A, B, and E. The administration of trivalent equine antitoxin to humans by the intravenous route neutralizes toxin molecules that are not yet bound to nerve endings. Before 1996, two to four 10-mL vials were administered to each adult patient suspected of having botulism; however, one vial (7500 IU of type A, 5500 IU of type B, and 8500 IU of type E antitoxins) per patient is now administered, and it is believed that no additional doses are necessary. Each vial contains an amount of antitoxin that is more than 100-fold greater than that needed to neutralize the largest amount of circulating antitoxin ever measured at the CDC. The circulating antitoxins have a half-life of 5 to 8 days, and a hypersensitivity reaction has been reported for up to 9% of patients. After the change to single-vial dosing, the incidence of hypersensitivity may be smaller than that previously reported. More than 80% of patients with adult infectious botulism in the United States are treated with antitoxin. The remaining 20% generally have such a prolonged delay in diagnosis that treatment is considered to be of no benefit; therefore, antitoxin is not administered. As of June 1998, this product is available in the United States solely for the treatment of infant botulism, under a Treatment Investigational New Drug protocol. For information on obtaining human botulism immune globulin, contact the California Department of Health Services at 510-540-2646 (24 hours)(Shapiro et al., 1998).
              • Contraindicator: Equine antitoxin therapy has not been recommended for infant botulism because of early observations (since disproved) that serum toxin was not detected in such cases and because of concerns about hypersensitivity reactions to this product(Shapiro et al., 1998).
              • Complication: Equine-derived antitoxin preparations have a number of possible side effects, including serum sickness and anaphylaxis, and are not recommended for use in infant botulism but do have a role in the treatment of food-borne and wound botulism in adults and noninfant children(Caya et al., 2004).
              • Success Rate: If it is administered early during the course of neurologic dysfunction, it is effective in preventing progression of illness and shortening the duration of ventilatory failure in severe cases of botulism. A retrospective analysis of 134 cases of type A botulism showed an overall mortality rate of 10% among patients who received early treatment with antitoxin (within 24 hours of symptom onset) compared with 15% among those who received late treatment (more than 24 hours after symptom onset) and 46% among those who did not receive antitoxin. In addition, survivors who received antitoxin early had a median hospital stay of only 10 days compared with 41 days for those who received antitoxin late and 56 days for those who did not receive antitoxin. The safety and efficacy of a human-derived antitoxin product (human botulism immune globulin) administered to infants with botulism are being determined(Shapiro et al., 1998).
            • Supportive care : Rapid diagnosis and the availability of well-coordinated, multidisciplinary supportive-care capabilities constitute the most effective measures in the treatment of botulism (whatever its type). Indeed, the keystone for supportive care is ventilatory support; protection and control of the airway by intubation and mechanical ventilation may not be necessary in mild cases of botulism, but they should be available and used whenever appropriate because these measures become an absolute necessity in cases of severe botulism. Although it is true that botulinum neurotoxins bind irreversibly to presynaptic endplates and irreparably impair these structures, axons possess a remarkable regenerative capability by which they can sprout new endplates if the patient can be successfully shepherded through the acute botulism crisis. Thus, supportive care is generally necessary for several days to several weeks until the patient is capable of survival without these measures. Antibiotics and debridement have very limited but nonetheless bona fide roles, principally in botulism of the wound type, to treat infection and clean up an injury site(Caya et al., 2004).
              • Contraindicator: Aminoglycosides, clindamycin, and polymyxin B should especially be eschewed because they have intrinsic neuromuscular blocking properties(Caya et al., 2004).
   4. Prevention:
      1. Botulism Prevention
         • Description: C. botulinum spores are ubiquitous. Safe food preservation methods destroy spores or inhibit their germination and growth. To reduce the risk for botulism when pickling, food items should be washed and cooked adequately, and utensils, containers, and other surfaces in contact with food, including cutting boards and hands, should be cleaned thoroughly with soap and warm water. Containers (e.g., jars and lids) in which pickling will occur should be sterilized (e.g., placed in boiling water for the prescribed period published in the container instructions). Adequate acidification to a pH less than or equal to 4.6 is essential. Refrigeration at 39 F (4 C) during pickling is advisable, especially in foods that may be acidified inadequately such as whole eggs. Once opened, any canned or pickled food should be refrigerated. Pricking, poking holes, or otherwise handling whole eggs in a manner that might allow spores or bacteria into the yolk should be avoided. When foodborne botulism is suspected, clinicians and public health investigators should inquire about the preparation and eating of foods preserved by any home method (e.g., canning, pickling, curing, and fermenting). Persons seeking advice on home-food preservation should consult their local county or university cooperative extension service, or contact the U.S. Department of Agriculture Food Safety Hotline, telephone (800) 535-4555. CDC provides epidemiologic consultation and laboratory diagnostic services for suspected botulism cases and authorizes release of botulism antitoxin. Through state health departments, these services are available 24 hours a day from CDC(CDC report, 2000), Although ingesting honey is a known risk factor, the source of spores for the majority of infant botulism cases is unknown. Ingestion of Clostridium botulinum spores, which exist worldwide in the soil and dust, is believed to be the principal route of exposure. Avoid feeding honey to infants aged less then 12 months. Report all cases to local and state health departments(CDC report, 2003),
   5. Model System:
      1. Human erythrocytes
         1. Model Host: .
            Binding of the progenitor toxins and recombinant proteins to erythrocytes was determined in a microassay(Fujinaga, 2000),
         2. Model Pathogens:
         3. Description: Microtiter plates (EIA/RIA Plate, Costar) were coated with the toxins or recombinant proteins (100 mg/ml, 50 ml per well) at 4C for 16 h. The wells were washed twice with PBS, and free binding sites were blocked with 1% bovine serum albumin (BSA) in PBS at room temperature for 2 h. Then the wells were incubated with a 1% suspension of human type O erythrocytes in PBS containing 1% BSA at room temperature for 30 min. The wells were carefully washed five times with PBS, the adherent erythrocytes were lysed with H₂O (50 microliters per well), and an absorbance at 405 nm of the hemoglobin was taken as a measure of the number of adherent erythrocytes(Fujinaga, 2000),
      1. Guinea pigs
         1. Model Host: .
            Cavia. Guinea pigs(Website 9),
         2. Model Pathogens:
         3. Description: Binding experiments of the progenitor toxins and recombinant proteins to paraformaldehyde-fixed sections of guinea pig upper small intestine(Fujinaga, 2000), Binding experiments were performed using intestinal tissue sections. Type A HA(hemagglutinin)-positive progenitor toxin (a mixture of 16S and 19S toxins) showed intense binding to the microvilli, whereas HA-negative progenitor toxin (12S toxin) did not show any binding, as reported for type C progenitor toxins. Given that type C 16S toxin exhibits NeuAc (N-acetylneuraminic acid)-dependent binding activity, the ability of type A HA-positive progenitor toxin to adhere to sialidase-treated intestinal sections was tested. The sialidase treatment showed no change in type A HA-positive progenitor toxin binding. In order to examine ligand(s) for type A HA-positive progenitor toxin on microvilli, binding inhibition experiments using three different saccharides were carried out. Binding was drastically inhibited by 100 mM lactose (Lac) and 100 mM galactose (Gal), but not by 100 mM glucose (Glc). These results indicate that the HA but not NTNH (non-toxic non-HA) or NTX (neurotoxin), components of type A HA-positive progenitor toxin exhibit binding activity on microvilli, as observed for type C. However, unlike type C HA, the Gal moieties on the microvilli glycoconjugates mediate toxin binding(Fujinaga, 2000),
      1. Mice
         1. Model Host: .
            Mus sp. Mice(Website 10),
         2. Model Pathogens:
         3. Description: Mice were injected intragastrically with 100000 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 post-challenge 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(Sugiyama and Mills, 1978),
      1. Mice
         1. Model Host: .
            Mus sp. Mice(Website 10),
         2. Model Pathogens:
         3. Description: An animal model of wound botulism was developed in mice using an inoculum of Clostridium botulinum type A spores. The number of C. botulinum in infected wounds was quantitated by culturing on egg yolk agar, and the level of C. botulinum toxin in infected wound tissue was measured by a bioassay in mice and by an enzyme-linked immunosorbent assay. All infected mice receiving no further treatment developed neuroparalytic symptoms consistent with botulism after an incubation period of ca. 48 h, and all of these animals died. Serotherapy with C. botulinum type A antitoxin initiated 24 h post-challenge reduced the mortality rate to 5%. Treatment with metronidazole 2 to 24 h post-challenge resulted in recovery rates of 40 to 91%(Dezfulian and Bartlett, 1985),
      1. Rats
         1. Model Host: .
            Rattus sp. Rats(Website 11),
         2. Model Pathogens:
         3. Description: A rat model of localized disuse induced by the Clostridium botulinum toxin(Chappard, 2001), Twelve male Wistar rats, aged between 19 and 20 weeks and weighing 506 57 g were used in experiments of generalized paralyzing disease caused by the botulinum toxin. Six rats received a 2-U injection of toxin in the right quadriceps. Six rats were similarly injected with saline and were used as control. The toxin acts 34 days after injection by blocking the release of acetylcholine to the muscle. Paralysis of the quadriceps was obtained 45 days after the injection(Chappard, 2001),
      1. Rats
         1. Model Host: .
            Rattus sp. Rats(Website 11),
         2. Model Pathogens:
         3. Description: The rat as an animal model for infant botulism(Moberg and Sugiyama, 1980), Only animals from 7 to less than 13 days old at time of intragastricchallenge with 10(5) spores were colonized; 9-dayold rats were the most susceptible. The colonized infants did not become ill, but C. botulinum toxin was present in the cecum-colon segment from 1 to 6 days postchallenge. The highest incidence of positives was obtained when the animals were tested for toxin on day 4 after administration of the inoculum. The mean infective dose for 9-day-old rats (1,500 spores per animal with 95% confidence limits of 1,300 to 1,800) was not significantly different from that for infant mice of the most susceptible age (700 spores with 170 to 3,000 confidence limits). Susceptibility to intraintestinal Clostridium botulinum colonization of conventional infant and germfree adult rats is comparable to that of mice. C. botulinum-monoassociated rats pass C. botulinum toxin in their milk(Moberg and Sugiyama, 1980),

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Arnon et al., 2001: Arnon SS, Schechter R, Inglesby TV, Henderson DA, Bartlett JG, Ascher MS, Eitzen E, Fine AD, Hauer J, Layton M, Lillibridge S, Osterholm MT, O'Toole T, Parker G, Perl TM, Russell PK, Swerdlow DL, Tonat K. Botulinum toxin as a biological weapon: medical and public health management. The Journal of the American Medical Association. 2001; 285(8); 1059-1070. [PubMed: 11209178].

CDC report, 2000: CDC . From the Center for Disease Control and Prevention. Foodborne botulism from eating home-pickled eggs--Illinois, 1997. JAMA : The Journal of the American Medical Association. 2000; 284(17); 2181-2182. [PubMed: 11184244].

CDC report, 2003: CDC . From the Centers for Disease Control and Prevention. Infant botulism--New York City, 2001-2002. JAMA : The Journal of the American Medical Association. 2003; 289(7); 834-836. [PubMed: 12599363].

Caya et al., 2004: Caya JG, Agni R, Miller JE. Clostridium botulinum and the clinical laboratorian: a detailed review of botulism, including biological warfare ramifications of botulinum toxin. Archives of Pathology and Laboratory Medicine. 2004; 128(6); 653-662. [PubMed: 15163234].

Chappard, 2001: Chappard D, Chennebault A, Moreau M, Legrand E, Audran M, Basle MF. Texture analysis of X-ray radiographs is a more reliable descriptor of bone loss than mineral content in a rat model of localized disuse induced by the Clostridium botulinum toxin. Bone. 2001; 28(1); 72-79. [PubMed: 11165945].

Cherington, 1998: Cherington M. Clinical spectrum of botulism. Muscle Nerve. 1998; 21(6); 701-710. [PubMed: 9585323].

Dahlenborg et al., 2001: Dahlenborg M, Borch E, Radstrom P. Development of a combined selection and enrichment PCR procedure for Clostridium botulinum Types B, E, and F and its use to determine prevalence in fecal samples from slaughtered pigs. Applied and Environmental Microbiology. 2001; 67(10); 4781-4788. [PubMed: 11571185].

Dahlenborg et al., 2003: Dahlenborg M, Borch E, Radstrom P. Prevalence of Clostridium botulinum types B, E and F in faecal samples from Swedish cattle. International Journal of Food Microbiology. 2003; 82(2); 105-110. [PubMed: 12568750].

Dezfulian and Bartlett, 1985: Dezfulian M, Bartlett JG. Kinetics of growth and toxigenicity of Clostridium botulinum in experimental wound botulism. Infection and Immunity. 1985; 49(2); 452-454. [PubMed: 3894236].

Ferreira et al., 2003: Ferreira JL, Maslanka S, Johnson E, Goodnough M. Detection of botulinal neurotoxins A, B, E, and F by amplified enzyme-linked immunosorbent assay: collaborative study. The Journal of AOAC International. 2003; 86(2); 314-331. [PubMed: 12723917].

Fujinaga, 2000: Fujinaga Y, Inoue K, Nomura T, Sasaki J, Marvaud JC, Popoff MR, Kozaki S, Oguma K. Identification and characterization of functional subunits of Clostridium botulinum type A progenitor toxin involved in binding to intestinal microvilli and erythrocytes. FEBS Letters. 2000; 467(2-3); 179-183. [PubMed: 10675534].

Moberg and Sugiyama, 1980: Moberg LJ, Sugiyama H. The rat as an animal model for infant botulism. Infection and Immunity. 1980; 29(2); 819-821. [PubMed: 7011988].

Passaro et al., 1998: Passaro DJ, Werner SB, McGee J, Mac Kenzie WR, Vugia DJ. Wound botulism associated with black tar heroin among injecting drug users. JAMA : The Journal of the American Medical Association. 1998; 279(11); 859-863. [PubMed: 9516001].

Plowman and Peck, 2002: Plowman J, Peck MW. Use of a novel method to characterize the response of spores of non-proteolytic Clostridium botulinum types B, E and F to a wide range of germinants and conditions. Journal of Applied Microbiology. 2002; 92(4); 681-694. [PubMed: 11966909].

Shapiro et al., 1998: Shapiro RL, Hatheway C, Swerdlow DL. Botulism in the United States: a clinical and epidemiologic review. Annals of Internal Medicine. 1998; 129(3); 221-228. [PubMed: 9696731].

Sobel et al., 2004: Sobel J, Tucker N, Sulka A, McLaughlin J, Maslanka S. Foodborne botulism in the United States, 1990-2000. Emerging Infectious Diseases. 2004; 10(9); 1606-1611. [PubMed: 15498163].

Sugiyama and Mills, 1978: Sugiyama H, Mills DC. Intraintestinal toxin in infant mice challenged intragastrically with Clostridium botulinum spores. Infection and Immunity. 1978; 21(1); 59-63. [PubMed: 361570].

Tanzi and Gabay, 2002: Tanzi MG, Gabay MP. Association between honey consumption and infant botulism. Pharmacotherapy. 2002; 22(11); 1479-1483. [PubMed: 12432974].

Website 10: NCBI. Taxonomy. Mice

Website 11: NCBI. Taxonomy. Rats

Website 9: NCBI. Taxonomy. Guinea pigs

Wu et al., 2001: Wu HC, Huang YL, Lai SC, Huang YY, Shaio MF. Detection of Clostridium botulinum neurotoxin type A using immuno-PCR. Letters in Applied Microbiology. 2001; 32(5); 321-325. [PubMed: 11328498].

 

PathInfo: George Abramochkin

HazARD: (for the section of Lab Animal Pathobiology & Management)

PHIDIAS: Yongqun "Oliver" He

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