Designing a field trial of an equine grass sickness vaccine: A questionnaire-based feasibility study.

Without an experimental model of equine grass sickness (EGS), a randomised controlled field trial (RCT) represents the only method of evaluating the efficacy of Clostridium botulinum type C vaccination in preventing naturally occurring disease. Clinical trial feasibility is an important aspect of preliminary work undertaken prior to initiating RCTs, estimating parameters that are important for study design. This cross-sectional study aimed to assess the feasibility of conducting a nationwide RCT of a candidate vaccine for EGS based on responses from a sample of British equine veterinary practices (n = 119/284). Seventy-three percent of practices had attended ≥1 EGS case within the preceding 2 years (median four cases), and 51.3% regularly attended recurrently affected premises. Veterinary surgeons had greater confidence diagnosing acute/subacute EGS based solely on history and clinical signs compared to chronic EGS. Ninety-one percent of respondents (n = 103/113) considered the proposed RCT to be important/very important to equine veterinary research. Ninety-one percent of respondents (n = 102/112) indicated preparedness to assist in owner recruitment and 92.9% (n = 104/112) indicated willingness to participate in a RCT. The most frequent reasons for practices declining to participate were low incidence of EGS (n = 4), did not believe clients would wish to participate (n = 3) and amount of paperwork/data collection involved (n = 2). There was considerable support amongst participating veterinary practices for a RCT evaluating the efficacy of Clostridium botulinum vaccination for the prevention of EGS in Britain. Substantial proportions of participating practices would be prepared to participate in the RCT and regularly attended EGS-affected premises that would meet trial inclusion criteria.

Production and evaluation of a recombinant chimeric vaccine against clostridium botulinum neurotoxin types C and D.

Bovine botulism is a fatal disease that is caused by botulinum neurotoxins (BoNTs) produced by Clostridium botulinum serotypes C and D and that causes great economic losses, with nearly 100% lethality during outbreaks. It has also been considered a potential source of human food-borne illness in many countries. Vaccination has been reported to be the most effective way to control bovine botulism. However, the commercially available toxoid-based vaccines are difficult and hazardous to produce. Neutralizing antibodies targeted against the C-terminal fragment of the BoNT heavy chain (HC) are known to confer efficient protection against lethal doses of BoNTs. In this study, a novel recombinant chimera, consisting of Escherichia coli heat-labile enterotoxin B subunit (LTB), a strong adjuvant of the humoral immune response, fused to the HC of BoNT serotypes C and D, was produced in E. coli. Mice vaccinated with the chimera containing LTB and an equivalent molar ratio of the chimera without LTB plus aluminum hydroxide (Al(OH)3) developed 2 IU/mL of antitoxins for both serotypes. Guinea pigs immunized with the recombinant chimera with LTB plus Al(OH)3 developed a protective immune response against both BoNT/C (5 IU/mL) and BoNT/D (10 IU/mL), as determined by a mouse neutralization bioassay with pooled sera. The results achieved with guinea pig sera fulfilled the requirements of commercial vaccines for prevention of botulism, as determined by the Brazilian Ministry of Agriculture, Livestock and Food, Supply. The presence of LTB was essential for the development of a strong humoral immune response, as it acted in synergism with Al(OH)3. Thus, the vaccine described in this study is a strong candidate for the control of botulism in cattle.

Production of serotype C specific and serotype C/D generic monoclonal antibodies using recombinant H(C) and H(N) fragments from Clostridium botulinum neurotoxin types C(1) and D.

Sequence variability of Clostridium botulinum serotypes C and D is particularly complex. Some serotype C and D strains have unique gene structures that encode mosaic isoforms of botulinum neurotoxin (BoNT) containing components of both BoNT type C(1) (BoNT/C(1)) and BoNT type D (BoNT/D). Such sequence variability and the potential for cross neutralisation must be taken into consideration when developing serotype C and D detection and identification assays. Three fusion proteins containing either a fragment from the carboxyl-terminal domain of the heavy chain (H(C)) of BoNT/C(1) (strain 573), a fragment from the H(C) of BoNT/D (strain BVD/-3) or a fragment from the amino-terminal domain of the heavy chain (H(N)) of BoNT/C(1) (strain 573) were expressed in Escherichia coli, and administered as immunogens to mice. Monoclonal antibodies (mAbs) against the recombinant BoNT fragments were prepared by three fusions. MAbs recognising native BoNT/C(1) and BoNT/D were detected by enzyme-linked immunosorbent assay (ELISA). Nine monoclonal antibodies (mAbs) were produced, six of which recognised a BoNT fragment that is highly conserved across all serotype C and D producing strains. We conclude that these mAbs and this approach to mAb production may facilitate the development of immunological diagnostic techniques that are not constrained by the existence of mosaic isoforms for the detection and identification of serotypes C and D.

Correlation of Clostridium botulinum type C antitoxin titers in mink and guinea pigs to protection against type C intoxication in mink.

In USA, the potency of commercial vaccines containing Clostridium botulinum type C toxoid is determined by a mink vaccination-challenge assay outlined in the Code of Federal Regulations, Title 9, Part 113.110. A more humane potency test is desired, and this study provides preliminary data in support of a serological assay that correlates post-vaccination antitoxin titers of guinea pigs to vaccine efficacy in mink. Mink and guinea pigs were injected with varying dilutions of a vaccine containing C. botulinum type C toxoid. Blood samples were collected from each animal prior to challenging the mink with type C toxin. Serum antitoxin titers of mink and guinea pigs were measured by a mouse protection test, and the results were compared to the outcome of the toxin challenge in mink. A dose-dependent antitoxin response was observed in guinea pigs vaccinated with the critical dilutions of vaccine bracketing the minimum protective dose in mink. These preliminary data suggest that it may be possible to correlate post-vaccination antitoxin titers in guinea pigs to vaccine efficacy in mink. This correlation could be used as the basis for a more humane potency test for C. botulinum type C toxoids.

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.

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.