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: