• Vaccine Ontology ID: VO_0004074
• Type: Subunit vaccine
• SCFV single-chain Fv fragment gene engineering:
  • Type: Protein
  • Detailed Gene Information: Click Here.
• Adjuvant:
  • VO ID: VO_0001241
  • Description: The botulinum neurotoxins (BoNT) are the causative agents of botulism and represent a family of seven structurally similar but antigenically distinct serotypes (A to G). These toxins exert their action by blocking release of the neurotransmitter acetylcholine at the neuromuscular junction. BoNT are usually expressed in as a single polypeptide chain and then post-translationally nicked, forming a dichain consisting of a 100-kDa heavy chain and a 50-kDa light chain held together by a single disulfide bond. Topologically, these neurotoxins are composed of three domains: binding, translocation, and catalytic, each of which is believed to play a role in intoxication. The carboxy-terminal portion of the heavy chain is responsible for binding nerve cell receptor(s). After toxin binding, it is thought to be internalized into an endosome through receptor-mediated endocytosis. It is believed that the 50-kDa amino-terminal domain of the heavy chain possesses channel-forming capabilities when in the acidic environment of the endosome, allowing internalization of the toxin. The final step in the mechanism involves zinc-dependent proteolysis by the catalytic domain of key cytosolic substrates necessary for neurotransmitter release (Byrne et al., 1998).
• Preparation: After fermentation and cell disruption, BoNT/A(Hc) was purified by using a three-step chromatographic process consisting of expanded-bed chromatography, Mono S cation-exchange chromatography, and hydrophobic interaction chromatography (Byrne et al., 1998).
• Virulence: (Byrne et al., 1998)
• Description: The botulinum neurotoxins (BoNT) are the causative agents of botulism and represent a family of seven structurally similar but antigenically distinct serotypes (A to G). These toxins exert their action by blocking release of the neurotransmitter acetylcholine at the neuromuscular junction. BoNT are usually expressed in as a single polypeptide chain and then post-translationally nicked, forming a dichain consisting of a 100-kDa heavy chain and a 50-kDa light chain held together by a single disulfide bond. Topologically, these neurotoxins are composed of three domains: binding, translocation, and catalytic, each of which is believed to play a role in intoxication. The carboxy-terminal portion of the heavy chain is responsible for binding nerve cell receptor(s). After toxin binding, it is thought to be internalized into an endosome through receptor-mediated endocytosis. It is believed that the 50-kDa amino-terminal domain of the heavy chain possesses channel-forming capabilities when in the acidic environment of the endosome, allowing internalization of the toxin. The final step in the mechanism involves zinc-dependent proteolysis by the catalytic domain of key cytosolic substrates necessary for neurotransmitter release (Byrne et al., 1998).

• Vaccine Ontology ID: VO_0004076
• Type: Toxoid vaccine
• Adjuvant:
  • VO ID: VO_0000127
  • Description: separate monovalent toxoid vaccine against BoNTF was manufactured for the U.S. Army by Porton Products Limited in cooperation with the United Kingdom Governments Center for Applied Microbiology and Research (CAMR) in 1990 (Byrne et al., 2000).
• Preparation: The vaccine batch no. 002/90 was derived from pooling three production lots of C. botulinum F toxin. The harvested toxin (i.e. by acid precipitation, tangential flow filtration, and centrifugation) from each of three fermentation runs was pooled and the type F toxin extracted with sodium phosphate buffer (PBS). After ribonuclease treatment, the toxin was further purified by ammonium sulfate precipitation, and repeated fractionation on fast liquid column chromatography on a fast flow Q Sepharose column. Unlike the toxoid (estimated toxoid purity of 10%), the purity of the type F botulinum toxoid Lot no. 002/90 was greater than 60% (IND 5077). The partially purified F toxin was formalin-detoxified and adsorbed to adjuvant (Byrne et al., 2000).
• Virulence: Even though toxoid vaccines are available, there are numerous shortcomings with their current use and ease of production. First, because C. botulinum is a spore-former, a dedicated facility is required to manufacture a toxin-based product. The requirement for a dedicated manufacturing facility is not trivial. It is extremely costly to renovate and upgrade an existing facility or to build a new one and then to maintain the facility in accordance with current Good Manufacturing Practices (cGMP) to manufacture one vaccine. Second, the yields of toxin production from C. botulinum are relatively low. Third, the toxoid-producing process involves handling large quantities of toxin and thus is dangerous, and the added safety precautions increase the cost of manufacturing. Fourth, the toxoid product for types A-E consists of a crude extract of clostridial proteins that may influence immunogenicity or reactivity of the vaccine, and the type F toxoid is only partially purified (IND 5077). Fifth, because the toxoid-producing process involves the use of formaldehyde, which inactivates the toxin, and residual levels of formaldehyde (not to exceed 0.02%) are part of the product formulation to prevent reactivation of the toxin, the vaccine is reactogenic. An additional component of the toxoid vaccines is the preservative thimerosal (0.01%), which also increases the reactogenicity of the product (Byrne et al., 2000).
• Description: A separate monovalent toxoid vaccine against BoNTF was manufactured for the U.S. Army by Porton Products Limited in cooperation with the United Kingdom Governments Center for Applied Microbiology and Research (CAMR) in 1990 (Byrne et al., 2000).

• Vaccine Ontology ID: VO_0004166
• Type: DNA vaccine
• Status: Research
• Host Species as Laboratory Animal Model: Mouse
• Antigen: Clostridium botulinum neurotoxin serotypes A (BoNT/A) and B (BoNT/B) (Yu et al., 2009)
• BoNT/B gene engineering:
  • Type: DNA vaccine construction
  • Detailed Gene Information: Click Here.
• BoNT/A1 from C. botulinum A str. Hall gene engineering:
  • Type: DNA vaccine construction
  • Detailed Gene Information: Click Here.
• Vector: pSCAR DNA replicon expression vector (Yu et al., 2009)
• Immunization Route: Intramuscular injection (i.m.)

• Vaccine Ontology ID: VO_0011504
• Type: Subunit vaccine
• Status: Research
• Antigen: C. botulinum FHc
• FHc gene engineering:
  • Type: Recombinant protein preparation
  • Description: A new gene encoding the Hc domain of Clostridium botulinum neurotoxin serotype F (FHc) was designed and completely synthesized with oligonucleotides. A soluble recombinant Hc of C. botulinum neurotoxin serotype F was highly expressed in Escherichia coli with this synthetic FHc gene (Yu et al., 2008).
  • Detailed Gene Information: Click Here.
• Adjuvant:
  • VO ID: VO_0000139
• Immunization Route: Subcutaneous injection

• Vaccine Ontology ID: VO_0004084
• Type: Subunit vaccine
• Adjuvant:
  • VO ID: VO_0000127
  • Description: Type C and D toxins provoke botulism in many animal species, including birds. C. botulinum type C organisms have been isolated from the contents of the gastric tract of the carcass and environmental materials such as soil, maggots, food, and/or straw mats. At present, the most widely available vaccine for humans and animals is formalin-inactivated toxoids. Although these are very effective, they are expensive and time-consuming to prepare and are ssomewhat hazardous during detoxification. To solve these problems, a recombinant vaccine has been considered (Arimitsu et al., 2004).
• Preparation: The C. botulinum type C strain, C-Stockholm (C-St), and the type D strain, D-1873, were used for the production and purification of type C and D toxins, respectively (Arimitsu et al., 2004).
• Virulence: (Arimitsu et al., 2004)
• Description: Type C and D toxins provoke botulism in many animal species, including birds. C. botulinum type C organisms have been isolated from the contents of the gastric tract of the carcass and environmental materials such as soil, maggots, food, and/or straw mats. At present, the most widely available vaccine for humans and animals is formalin-inactivated toxoids. Although these are very effective, they are expensive and time-consuming to prepare and are ssomewhat hazardous during detoxification. To solve these problems, a recombinant vaccine has been considered (Arimitsu et al., 2004).

• Vaccine Ontology ID: VO_0004099
• Type: DNA vaccine
• Adjuvant:
  • VO ID: VO_0001241
  • Description: Previous research achieved protection in a murine model using purified FHc which had been obtained from cultures of recombinant Escherichia coli and recombinant Pichia pastoris. FHc has also been expressed in a Salmonella vector and achieved protection against intoxication in a murine model. Therefore, BoNT F could constitute a good candidate for DNA vaccination, and the given study constructed a DNA vaccine based on the Hc domain of BoNT subtype F in order to investigate the utility of DNA vaccination for protection against intoxication with this subtype (Bennett et al., 2003).
• Vector: pSecTag2C (Bennett et al., 2003)
• Preparation: DNA encoding the binding domain of BoNT subtype F (FHc) was obtained as the plasmid clone pHILD4.New.FHc. Recombinant plasmid DNA was purified using Endofree Mega-Q kits (Qiagen Ltd.). Cells were analysed for expression of FHc 24 h after transfection as follows: cells were fixed in 4% paraformaldehyde at 4 °C overnight, then washed in PBS and incubated in PBS containing 2% saponin and 10% foetal calf serum (PBS-SFCS) for 2 h at 37 °C. Primary antibody, from mice immunised with pABFHc2 and boosted with recombinant FHc protein, was diluted 1:500 in PBS-SFCS and incubated with fixed cells for 1 h at 37 °C. Following washing, anti-mouse IgG conjugated to fluorescein isothiocyanate was added and the incubation was continued for a further hour at 37 °C. The cells were washed in PBS and were visualised by fluorescent confocal microscopy (Bennett et al., 2003).
• Description: Previous research achieved protection in a murine model using purified FHc which had been obtained from cultures of recombinant Escherichia coli and recombinant Pichia pastoris. FHc has also been expressed in a Salmonella vector and achieved protection against intoxication in a murine model. Therefore, BoNT F could constitute a good candidate for DNA vaccination, and the given study constructed a DNA vaccine based on the Hc domain of BoNT subtype F in order to investigate the utility of DNA vaccination for protection against intoxication with this subtype (Bennett et al., 2003).

• Vaccine Ontology ID: VO_0004078
• Type: Recombinant vector vaccine
• Preparation: Construction begins with the VEE replicon vector, the capsid helper, and the glycoprotein helper, which contains attenuating mutations. Construction, characterization, and assembly of the replicons into VRP for the BoNT/A HC, the anthrax MAT-PA replicon, the MBGV-GP replicon, and the negative control comprising mutagenized staphylococcal enterotoxin B (mSEB) replicon were then prepared. The BoNT/C HC gene was PCR-cloned into the VEE replicon plasmid by using Cla I restriction enzyme recognition sequence—gene specific primers. The VRP titers are expressed as focus-forming units (FFU) where 1 FFU is equivalent to 1 infectious unit (iu) (Lee et al., 2006).
• Virulence: (Lee et al., 2006)
• Description: The development of multi-agent vaccines offers the advantage of eliciting protection against multiple diseases with minimal inoculations over a shorter time span. Formulations consisted of individual Venezuelan equine encephalitis (VEE) virus replicon-vectored vaccines against a bacterial disease (anthrax), a viral disease (Marburg fever), and a toxin-mediated disease (botulism). The individual VEE replicon particles (VRP) expressed mature 83-kDa protective antigen (MAT-PA) from Bacillus anthracis, the glycoprotein (GP) from Marburg virus (MBGV), or the H(C) fragment from botulinum neurotoxin (BoNT H(C)) (Lee et al., 2006).

Mouse Response

• Host Strain: Cr1:CD-1 (ICR) mice (Charles River).
• Vaccination Protocol: Each of 10 mice per group was injected one to three times with 0.01, 0.1, 0.5, 1.0, or 2.0 µg FPLC-purified BoNT/A(Hc). Multiple injections were given 14 days apart. Two days before challenge, mice were bled retro-orbitally for ELISA and serum neutralization testing (Byrne et al., 1998).
• Persistence: ELISA titers for individual mice successfully predicted survival. When the titers were at least 1600, 98.8% of the mice survived. When the titers were 100 or less, mice had only a 14.3% survival rate (Byrne et al., 1998).
• Side Effects: None noted (Byrne et al., 1998).
• Challenge Protocol: Mice were challenged 21 days after the last injection with 1,000 mouse i.p. LD50 of BoNT/A toxin complex diluted in GPB in a total volume of 100 µl per mouse. Mice were observed daily, and deaths were recorded 5 days post-challenge (Byrne et al., 1998).
• Efficacy: In general, multiple injections protected better than one, with complete or nearly complete protection realized at doses of 0.5 µg/mouse (Byrne et al., 1998).
• Description: Inhibition of BoNT action at a key step of the process could abolish the onset of botulism. One approach to developing a vaccine against botulism would be to construct and express a gene encoding only the binding domain of BoNT [BoNT(Hc)] and purify the translated product. This material, when administered to an organism, would not cause botulism because it lacks the enzyme and should not be able to enter the nerve cell without the translocation domain. Antibodies toward the product which neutralize BoNT serotype A (BoNT/A) toxicity when the host is directly challenged could be produced. Currently, a toxoid vaccine against BoNT serotypes A to E is used. However, there are inherent problems with the toxoid. The product consists of a crude extract of clostridial proteins. The material is dangerous to produce, and there is a high cost associated with preparing the toxoid vaccine. The toxoid also contains formalin, which is very painful for the recipient. Finally, only five of the seven serotypes are represented in the formulation. Thus, the aim of the present work was to develop a process for isolating a highly immunogenic recombinant BoNT(Hc) which could protect animals against a direct challenge of BoNT and that would be cheaper and less dangerous to produce. Ultimately, the developed process will be licensed as a vaccine (Byrne et al., 1998).

Mouse Response

• Host Strain: BALB/c
• Vaccination Protocol: Mice were injected with 50 μl inoculum into each hind leg of each mouse. All mice were tail bled for sera 1 day prior to toxin challenge. Mice were then challenged i.p. with 10^4 MLD of type F C. botulinum toxin (BoNT/F). Mice were challenged 28 d after their final vaccination. Untreated groups of age-matched mice were used as controls (Jathoul et al., 2004).
• Persistence: (Jathoul et al., 2004)
• Side Effects: None were noted (Jathoul et al., 2004).
• Efficacy: BoNT/F DNA vaccine provided 90% protection against 10^4 MLD BoNT/F in mice following two immunizations with 100 μg DNA (Jathoul et al., 2004).
• Description: A vaccine consisting of inactivated C. botulinum culture supernatants has been produced to provide military personnel with protection against BoNTs A, B, C, D and E. However, disadvantages associated with this vaccine include its expense and requirement for frequent boosters to maintain protection, as well as its efficacy against only 5 of the 7 BoNT types. Thus, new generation botulinum vaccines are being investigated, including the use of non-toxic BoNT Hc fragments as antigens. More recently, DNA vaccines encoding BoNT/A Hc and type F botulinum neurotoxin Hc fragment (BoNT/F Hc) have been evaluated as candidate next generation botulism vaccines. Such DNA vaccines may be optimized in terms of the regulatory elements used to drive expression of BoNT Hc (Jathoul et al., 2004).

Mouse Response

• Vaccine Immune Response Type: VO_0003057
• Efficacy: Vaccinated mice were challenged with doses of 1000 and 10,000 LD50 of BoNT/A and BoNT/B, and immune protection was observed. 100% of mice (8/8) survived in the group challenged with 1000 LD50, which was significantly higher protection than that of the single pSCARSAHc or pSCARSBHc-vaccinated mice groups, and 87.5% of mice (7/8) survived in the group challenged with 10,000 LD50 (Yu et al., 2009).

Mouse Response

• Host Strain: BALB/c
• Vaccination Protocol: Female BALB/c mice (specific pathogen free), 6 weeks of age, were randomly assigned to different groups. In the first vaccination study, groups of six mice were vaccinated with 1 or 10 μg of FHc. The FHc protein in phosphate-buffered saline (PBS) was mixed 50:50 with complete Freund adjuvant (Sigma) for the first vaccination and with incomplete Freund adjuvant (Sigma) for the second or third vaccinations. Each mouse was treated with 0.4 ml of the material in either two (days 0 and 14) or three vaccinations (days 0, 14, and 28) via the subcutaneous route. Each vaccination group was repeated once. In the second vaccination study, groups of six mice were vaccinated intramuscularly (i.e., in each thigh quadriceps bilaterally) with either one, two, or three doses of 0.04, 0.2, 1, or 5 μg of FHc. Vaccine was diluted in 25% (vol/vol) Alhydrogel (Sigma), and injections were administered at 3-week intervals (100 μl/injection). For a negative control, PBS instead of the antigen was mixed with the adjuvant. The mice were tail bled for sera before each immunization or neurotoxin challenge (Yu et al., 2008).
• Challenge Protocol: Mice were challenged intraperitoneally (i.p.) with 2 × 10^3 or 2 × 10^4 50% lethal doses (LD50) of BoNT/F (Langeland strain, from the National Institute for the Control of Pharmaceutical and Biological Products) 2 weeks after the last vaccination. The mice were observed for 1 week, and death or survival was recorded (Yu et al., 2008).
• Efficacy: Purified FHc was used to vaccinate mice and evaluate their survival against challenge with active botulinum neurotoxin serotype F (BoNT/F). Mice that received one injection of 5 microg or two injections of >or=0.04 microg of FHc were completely protected (Yu et al., 2008).

Mouse Response

• Host Strain: ddY
• Vaccination Protocol: Male 6- to 8-week-old ddY strain mice ,purchased from Shimizu Laboratory Supplies Co., Ltd. (Kyoto, Japan), were immunized according to protocol. As a negative control, PBS instead of the antigen was mixed with the adjuvant. Each antigen solution was injected s.c. into the dorsal side of the mice (0.1 ml). At 3 weeks post-immunization, a second immunization was performed. Partial bleeding was performed via the tail vein (mice) at 3 and 5 weeks after the primary immunization, and the specific antibody titers were checked by ELISA and Western blotting tests (Arimitsu et al., 2004).
• Persistence: (Arimitsu et al., 2004)
• Side Effects: None were noted (Arimitsu et al., 2004).
• Efficacy: The mice were challenged with lethal doses of the 16 S toxins. All 5 mice immunized with type C-H chain survived a 10^5 mouse i.p. MLD of C-16 S toxin with no symptoms. However, 4/6 mice challenged with a 10^6 mouse i.p. MLD died, and the 2 surviving mice showed severe botulism. On the other hand, all 5 mice immunized with type D-H chain were completely protected even though they were challenged with a 10^6 mouse i.p. MLD of D-16 S toxin. When the mice that survived the challenge with type C and D toxins were then cross-challenged with 10 mouse i.p. MLD of D and C toxins, respectively, no mice survived.
• Description: Since it appears to be difficult to prepare a large amount of recombinant whole neurotoxin, the study attempted to prepare recombinant HC containing the histidine (His) tag of types C and D, and the vaccine effects were analyzed in mice (Arimitsu et al., 2004).

Mouse Response

• Host Strain: Balb/c mice (female, 6–8-week-old).
• Vaccination Protocol: Mice were injected with 50 μl and received up to five vaccinations. For protein boosting of DNA-vaccinated mice, E. coli-derived MBP-FHc was formulated in alhydrogel (20% (v/v)). A single dose of 5 μg in a volume of 100 μl was administered by i.p. injection. Blood was taken from a tail vein 12 days after each injection for serum antibody analysis by enzyme-linked immunosorbent assay (ELISA). All mice were microchipped in order to monitor antibody response and survival of individual animals. Mice were challenged with a purified preparation containing 10^4 MLD of botulinum toxin serotype F by i.p. injection (Bennett et al., 2003).
• Side Effects: None noted.
• Efficacy: A minimum of three vaccinations with pABFHc2, given over a 4-week period, were sufficient to protect 100% of mice against the high challenge dose (10^4 MLD of BoNT type F). Two doses of pABFHc2 protected up to 90% of vaccinated animals. A single dose of pABFHc2 protected 60% of mice when challenged with toxin at least 28 days after vaccination (Bennett et al., 2003).
• Description: Vaccination with DNA encoding the Hc domain of botulinum neurotoxin subtype A has been attempted but had limited success; thus a clinically viable DNA vaccine for subtype A was not achieved. BoNT F is serologically distinct from A and the amino acid sequences of type F toxins from different strains of Clostridia fall into a distinct phylogenetic group. Type F toxin cleaves VAMP (vesicle associated membrane protein), whereas BoNT A cleaves SNAP-25. Protection in a murine model has been previously achieved using purified FHc which had been obtained from cultures of recombinant Escherichia coli and recombinant Pichia pastoris. FHc was also expressed in a Salmonella vector and achieved protection against intoxication in a murine model. Therefore, BoNT F could constitute a good candidate for DNA vaccination. A DNA vaccine was constructed based on the Hc domain of BoNT subtype F in order to investigate the utility of DNA vaccination for protection against intoxication with this subtype (Bennett et al., 2003).

Mouse Response

• Host Strain: Swiss, CBA/J
• Vaccination Protocol: Swiss and CBA/J mice were inoculated behind the neck with either a single VRP or with a mixture of VRPs at a dose of 10^7 iu of each VRP in 200 μl of PBS on days 0, 35, and 70. The mice were challenged i.p. on d 105 or 164 with 1000 50% median lethal doses (MLD50) of BoNT/A or BoNT/C diluted in PBS containing 0.2% gelatin, respectively. CBA/J mice were challenged s.c. on d 105 with 10 LD50 units (2 × 10^8 spores) of heat-shocked spores of the Sterne strain of B. anthracis. Positive control mice were inoculated with 0.1 ml of anthrax vaccine adsorbed (AVA, Bioport Corp., Lansing, MI) or with 0.1 ml of pentavalent botulinum toxoid vaccine, and negative control mice were inoculated with mSEB VRP, and all were used as controls for the challenges. As a comparison, the dose of AVA or toxoid vaccine administered to humans is 0.5 ml. Sera for ELISA were obtained 2 d before each inoculation and 2 d before challenge.
• Persistence: The quality (i.e. neutralizing verses non-neutralizing antibody activity) of the anti-C/HC antibody response was considerably better than the anti-A/HC antibody response measured for CBA/J mice or that the BoNT/C circulated in the animals for longer periods before entering neurons thus allowing more time for antibody-mediated neutralization of the toxin in the animal after challenge (Lee et al., 2006).
• Side Effects: No adverse reactions or side effects were noted in mice receiving individual or combination VRP vaccines (Lee et al., 2006).
• Efficacy: Inoculation of Swiss mice on d 0, 35, and 70 with 10^7 iu of BoNT/A HC VRP, either alone or in combinations with MAT-PA or MBGV-GP, or with all three VRP vaccines, completely protected the mice from challenge with BoNT/A. CBA/J mice inoculated with BoNT/A HC VRP were 90% protected from a BoNT/A challenge, whereas a mix of BoNT/A HC and MAT-PA VRP poorly protected the animals (2/10 survived) from the same BoNT/A challenge (Lee et al., 2006).
• Description: Increased concerns about the use of biological agents in acts of terrorism and warfare have also increased the need for the rapid development of vaccines against a wide range of bacteria, toxins, and viruses. The NIAID has classified biological organisms and toxins that could be used in bioterrorism and biowarfare as category A, B, or C priority pathogens. In response, current studies involve a multiagent vaccine that utilizes the VEE virus replicon as a vector for BoNT, anthrax, and MBGV, all of which are classified as category A pathogens (Lee et al., 2006).

Ducks Response

• Host Strain: The ducks were a cross of Japanese Mallard and Khaki Cambell, male and female, 3 weeks old, and were purchased from the Takahashi Hatching Farm (Osaka, Japan).
• Vaccination Protocol: Male and female 3-week-old ducks (a cross of Japanese Mallard and Khaki Cambell, purchased from the Takahashi Hatching Farm in Osaka, Japan) were immunized with 0.2 ml dorsal injections. As a negative control, PBS instead of the antigen was mixed with the adjuvant. At 3 weeks post-immunization, a second immunization was performed. Partial bleeding was performed via the basilic vein at 3 and 5 weeks after the primary immunization, and the specific antibody titers were checked by ELISA and Western blotting tests (Arimitsu et al., 2004).
• Persistence: (Arimitsu et al., 2004)
• Side Effects: None were noted (Arimitsu et al., 2004).
• Efficacy: All 7 immunized ducks resisted the challenge with 10 duck i.v. MLD, but the survival rate decreased to 5/7 (71.4%) and 4/7 (57.1%) when the birds were challenged with 10^2 and 10^3 duck i.v. MLD (Arimitsu et al., 2004).
• Description: Type C and D toxins provoke botulism in many animal species, including the avian form. 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. Young ducks are released into a rice field to exterminate harmful insects or unwanted plants, grow up during the rice crop, and are used as meats after the completion of the harvest. However, 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 GI tract of the carcass and environmental materials such as soil, maggots, food, and/or straw mats. A study of vaccination in ducks ensued (Arimitsu et al., 2004).