Environmental factors influencing the prevalence of a Clostridium botulinum type C/D mosaic strain in nonpermanent Mediterranean wetlands.

Between 1978 and 2008, 13 avian botulism outbreaks were recorded in the wetlands of Mancha Húmeda (central Spain). These outbreaks caused the deaths of around 20,000 birds from over 50 species, including globally endangered white-headed ducks (Oxyura leucoceophala). Here, a significant association was found between the number of dead birds recorded in each botulism outbreak and the mean temperature in July (always >26°C). The presence of Clostridium botulinum type C/D in wetland sediments was detected by real-time PCR (quantitative PCR [qPCR]) in 5.8% of 207 samples collected between 2005 and 2008. Low concentrations of Cl(-) and high organic matter content in sediments were significantly associated with the presence of C. botulinum. Seventy-five digestive tracts of birds found dead during botulism outbreaks were analyzed; C. botulinum was present in 38.7% of them. The prevalence of C. botulinum was 18.2% (n = 22 pools) in aquatic invertebrates (Chironomidae and Corixidae families) and 33.3% (n = 18 pools) in necrophagous invertebrates (Sarcophagidae and Calliphoridae families), including two pools of adult necrophagous flies collected around bird carcasses. The presence of the bacteria in the adult fly form opens up new perspectives in the epidemiology of avian botulism, since these flies may be transporting C. botulinum from one carcass to another.

Type C botulism in a commercial turkey farm: a case report.

Botulism is an intoxication caused by exotoxins of Clostridium botulinum. The case of botulism described here occurred on a commercial meat turkey farm with two houses. Toms and hens were maintained in two separate houses, toms in house A and hens in house B. At 10 wk of age, an increase in mortality was observed in the toms located in house A. Clinically the animals presented with paralysis of the legs, wings, and neck. Affected birds were sitting and reluctant to move. Necropsy failed to find any specific lesions. In liver, heart, muscles, crop, and gizzard as well as in intestinal contents, DNA of C. botulinum type C was detected by PCR. The result was confirmed by a mouse lethality neutralization test. During the 2 wk after the onset of the clinical signs the mortality was about 12%. The hens kept in house B did not show any symptoms and remained healthy. Investigations of environmental samples to detect the source of the toxin were not successful. After 2 wk clinical signs and mortality abated. At 16 wk of age, toms again showed the same clinical signs accompanied by raised mortality. Again C. botulinum toxin type C was detected. Within 2 wk the total mortality reached roughly 50%. Based on the "precautionary principle" and in agreement with the local authorities, the birds were euthanatized using CO2 in order to not compromise food safety.

Molecular diversity of the two sugar-binding sites of the ß-trefoil lectin HA33/C (HA1) from Clostridium botulinum type C neurotoxin.

A critical role in internalizing the Clostridium botulinum neurotoxin into gastrointestinal cells is played by nontoxic components complexed with the toxin. One of the components, a ß-trefoil lectin has been known as HA33 or HA1. The HA33 from C. botulinum type A (HA33/A) has been predicted to have a single sugar-binding site, while type C HA33 (HA33/C) has two sites. Here we constructed HA33/C mutants and evaluated the binding capacities of the individual sites through mucin-assay and isothermal titration calorimetry. The mutant W176A (site I knockout) had a K(d) value of 31.5mM for galactose (Gal) and 61.3mM for N-acetylgalactosamine (GalNAc), while the K(d) value for N-acetylneuraminic acid (Neu5Ac) was too high to be determined. In contrast, the double mutant N278A/Q279A (site II knockout) had a K(d) value of 11.8mM for Neu5Ac. We also determined the crystal structures of wild-type and the F179I mutant in complex with GalNAc at site II. The results suggest that site I of HA33/C is quite unique in that it mainly recognizes Neu5Ac, and site II seems less important for the lectin specificity. The architectures and the properties of the sugar-binding sites of HA33/C and HA33/A were shown to be drastically different.

Temperature dependency of Clostridium botulinum C and D toxin production from anaerobically enriched bovine gastrointestinal samples.

AIMS: To determine whether Clostridium botulinum neurotoxin (BoNT) production in anaerobic culture was affected by temperature and could influence the sandwich ELISA (sELISA) detection of group III toxins in pre-enriched gastrointestinal (GI) contents from clinically suspect cattle botulism cases. METHODS AND RESULTS: Bovine post-mortem GI samples taken from 124 and 96 animals with suspect and nonsuspect botulism, respectively, were pre-enriched anaerobically at 30 and 37°C prior to testing by sELISA. After enrichment at 37°C, BoNT was demonstrated in all clinically suspect bovine botulism cases that had been identified by the mouse bioassay, and enrichment by both temperatures enabled BoNT detection in a number of mouse bioassay-negative suspect cases. CONCLUSIONS: Culture temperature does influence the production of group III BoNT, and incubation at both 30 and 37°C is required for optimum detection. SIGNIFICANCE AND IMPACT OF THE STUDY: The in vitro assay defined in this study has the potential of improving the confirmation rate of clinically suspect cattle botulism cases whilst reducing the use of the costly and ethically sensitive mouse bioassay, the current diagnostic gold standard for BoNT testing.

Effect of nicking the C-terminal region of the Clostridium botulinum serotype D neurotoxin heavy chain on its toxicity and molecular properties.

A unique strain of Clostridium botulinum serotype D 4947 produces toxin complexes that are composed of un-nicked components, including a neurotoxin (BoNT) and auxiliary proteins. This BoNT showed aberrant elution upon Superdex gel filtration, indicating a much lower molecular weight, due to hydrophobic interaction with the column. Limited trypsin proteolysis of BoNT produces two nicks; first nick yielded a BoNT 50 kDa light chain disulfide linked to a 100 kDa heavy chain (Hc), and a second nick arose in Hc C-terminal 10 kDa. The second nick occurred in the putative binding domain of the BoNT molecule and induced alterations in its secondary structure, leading to a significant reduction of mouse toxicity in comparison with that of the fully-activated singly nicked BoNT. These results help to clarify the role of the C-terminal half of the Hc in the oral toxicity of single-chain and more complex forms of BoNT.

Seroprevalence of antibotulinum neurotoxin type C antibodies in horses in Israel.

REASONS FOR PERFORMING STUDY: Clostridium botulinum type C is prevalent in Israel and outbreaks recorded in many species, other than horses. Association between levels of anti-BoNT/C antibodies and equine grass sickness (EGS) have been demonstrated but seroprevalence of anti-BoNT/C antibodies in horses has not been reported nor has EGS been reported in Israel. OBJECTIVES: To determine the seroprevalence of specific anti-BoNT/C antibodies in horses in Israel and to determine whether age, breed and gender, or geographical region of farms are potential risk factors for exposure to BoNT/C. HYPOTHESIS: Anti-BoNT/C antibodies are prevalent among horses in Israel and farm and horse-level variables are associated with increased risk for exposure. METHODS: Serum samples from 198 horses were collected and the levels of specific anti-BoNT/C antibodies were determined using enzyme-linked immunosorbent assay (ELISA). For each categorical variable indicator variables were created and the odds ratio (OR) and 95% confidence intervals (95% CI) for the outcome variable were calculated using a univariable and multivariable logistic regression models. RESULTS: A total of 61 (30.8%) horses were ELISA positive for anti-BoNT/C IgG antibodies. The farm and its geographical region were associated significantly with seropositivity, horse-level variables, such as gender and breed, were also associated with seropositivity. Quarter Horse and Warmblood mares placed in the southern region of Israel had the highest odds to be tested positive for anti-BoNT/C IgG antibodies. CONCLUSIONS AND POTENTIAL RELEVANCE: Several farm and various horse-level risk factors for exposure to BoNT/C, found in this study, could be correlated to previously reported risk factors of EGS. Studies are required to determine the predisposing factors that cause EGS, which is apparently not present in Israel.

Presence of antibotulinum neurotoxin antibodies in selected wild canids in Israel.

Serum samples from 35 golden jackals (Canis aureus syriacus), eight wolves (Canis lupus), and four red foxes (Vulpes vulpes) from various regions of Israel were collected during the years 2001-04 and tested for antibodies to Clostridium botulinum neurotoxin (BoNT) types C and D. Antibodies against BoNT types C and D were detected in 10 (29%) and in 3 (9%) of 35 golden jackals, respectively, using enzyme-linked immunosorbent assay. This report describes detection of anti BoNT antibodies in wild canids other than coyotes (Canis latrans) for the first time and demonstrates that C. botulinum type C is prevalent in Israel.

Sugar-binding sites of the HA1 subcomponent of Clostridium botulinum type C progenitor toxin.

Clostridium botulinum type C 16S progenitor toxin contains a hemagglutinin (HA) subcomponent, designated HA1, which appears to play an important role in the effective internalization of the toxin in gastrointestinal epithelial cells and in creating a broad specificity for the oligosaccharide structure that corresponds to various targets. In this study, using the recombinant protein fused to glutathione S-transferase, we investigated the binding specificity of the HA1 subcomponent to sugars and estimated the binding sites of HA1 based on X-ray crystallography and soaking experiments using various sugars. N-Acetylneuraminic acid, N-acetylgalactosamine, and galactose effectively inhibited the binding that occurs between glutathione S-transferase-HA1 and mucins, whereas N-acetylglucosamine and glucose did not inhibit it. The crystal structures of HA1 complex with N-acetylneuraminic acid, N-acetylgalactosamine, and galactose were also determined. There are two sugar-binding sites, sites I and II. Site I corresponds to the electron densities noted for all sugars and is located at the C-terminal beta-trefoil domain, while site II corresponds to the electron densities noted only for galactose. An aromatic amino acid residue, Trp176, at site I has a stacking interaction with the hexose ring of the sugars. On the other hand, there is no aromatic residue at site II; thus, the interaction with galactose seems to be poor. The double mutant W176A at site I and D271F at site II has no avidity for N-acetylneuraminic acid but has avidity for galactose. In this report, the binding specificity of botulinum C16S toxin HA1 to various sugars is demonstrated based on its structural features.