Development and evaluation of indirect enzyme-linked immunosorbent assays for the determination of immune response to multiple clostridial antigens in vaccinated captive bred southern white rhinoceros (Ceratotherium simum simum).

BACKGROUND: An overall increase in poaching of white rhinoceros results in captive breeding becoming a significant component of white rhinoceros conservation. However, this type of conservation comes with its own difficulties. When wildlife is captured, transported and/or confined to a boma environment, they are more predisposed to diseases caused by bacterial organisms such as spore forming Clostridium spp. A southern white rhinoceros (Ceratotherium simum simum) population on a captive bred farm was suspected to be affected by Clostridium infections. These endangered animals were apparently exposed to Clostridium spp., in the conservation area previously used for cattle farming. The rhinoceros population on the breeding operation property was vaccinated with a multi-component clostridial vaccine registered for use in cattle. Multiple indirect enzyme-linked immunosorbent assays (iELISAs) were developed in order to evaluate the serum antibody titres of these vaccinated animals. In evaluating vaccine efficacy, the gold standard mouse neutralization test (MNT) was not available and therefore iELISAs were developed for the detection of serum antibodies to C. perfringens type A (alpha toxin), C. chauvoei (whole cell), C. novyi (alpha toxin), C. septicum (alpha toxin) and C. sordellii (lethal toxin) in the white rhinoceros population using international reference sera of equine origin. Antibody titres against each clostridial antigen was evaluated in the vaccinated white rhinoceros population (n = 75). Analytical specificity showed slight cross-reactions for C. chauvoei and C. perfringens type A with the other antigens. Individual assay cut-off values were calculated with 95% confidence. Coefficient of variance (CV) values for both the international reference sera and in-house control sera across all the antigens were well below 16%, indicating good assay repeatability. This convenient and fast assay is suitable for monitoring humoral immune responses to clostridial antigens in vaccinated white rhinoceroses. RESULTS: Checkerboard titrations indicated optimal antigen and antibody concentrations to be used for each respective iELISA developed. Each titration set of the respective international reference and in-house control sera showed good repeatability with low standard deviations and coefficient of variance values calculated between repeats for each antigen. Individual assays proved repeatable and showed good analytical sensitivity and specificity. CONCLUSIONS: The developed iELISAs are able to evaluate antibody profiles of phospholipase C, C. chauvoei whole cells, TcnA, ATX, TcsL in white rhinoceros serum using international reference sera.

Immunogenicity of anthrax recombinant peptides and killed spores in goats and protective efficacy of immune sera in A/J mouse model.

Anthrax is primarily recognized as an affliction of herbivores with incubation period ranging from three to five days post-infection. Currently, the Sterne live-spore vaccine is the only vaccine approved for control of the disease in susceptible animals. While largely effective, the Sterne vaccine has several problems including adverse reactions in sensitive species, ineffectiveness in active outbreaks and incompatibility with antibiotics. These can be surmounted with the advent of recombinant peptides (non-living) next generation vaccines. The candidate vaccine antigens comprised of recombinant protective antigen (PA), spore-specific antigen (bacillus collagen-like protein of anthracis, BclA) and formaldehyde inactivated spores (FIS). Presently, little information exists on the protectivity of these novel vaccine candidates in susceptible ruminants. Thus, this study sought to assess the immunogenicity of these vaccine candidates in goats and evaluate their protectivity using an in vivo mouse model. Goats receiving a combination of PA, BclA and FIS yielded the highest antibody and toxin neutralizing titres compared to recombinant peptides alone. This was also reflected in the passive immunization experiment whereby mice receiving immune sera from goats vaccinated with the antigen combination had higher survival post-challenge. In conclusion, the current data indicate promising potential for further development of non-living anthrax vaccines in ruminants.

Comparative analysis of the immunologic response induced by the Sterne 34F2 live spore Bacillus anthracis vaccine in a ruminant model.

The Sterne 34F2 live spore vaccine (SLSV) developed in 1937 is the most widely used veterinary vaccine against anthrax. However, literature on the immunogenicity of this vaccine in a target ruminant host is scarce. In this study, we evaluated the humoral response to the Bacillus anthracis protective antigen (rPA), a recombinant bacillus collagen-like protein of anthracis (rBclA), formaldehyde inactivated spores (FIS) prepared from strain 34F2 and a vegetative antigen formulation prepared from a capsule and toxin deficient strain (CDC 1014) in Boer goats. The toxin neutralizing ability of induced antibodies was evaluated using an in vitro toxin neutralization assay. The protection afforded by the vaccine was also assessed in vaccinates. Anti-rPA, anti-FIS and lethal toxin neutralizing titres were superior after booster vaccinations, compared to single vaccinations. Qualitative analysis of humoral responses to rPA, rBclA and FIS antigens revealed a preponderance of anti-FIS IgG titres following either single or double vaccinations with the SLSV. Antibodies against FIS and rPA both increased by 350 and 300-fold following revaccinations respectively. There was no response to rBclA following vaccinations with the SLSV. Toxin neutralizing titres increased by 80-fold after single vaccination and 700-fold following a double vaccination. Lethal challenge studies in naïve goats indicated a minimum infective dose of 36 B. anthracis spores. Single and double vaccination with the SLSV protected 4/5 and 3/3 of goats challenged with>800 spores respectively. An early booster vaccination following the first immunization is suggested in order to achieve a robust immunity. Results from this study indicate that this crucial second vaccination can be administered as early as 3 months after the initial vaccination.

Development of a flow cytometric bead immunoassay and its assessment as a possible aid to potency evaluation of enterotoxaemia vaccines.

Enterotoxaemia, an economically important disease of sheep, goats and calves, is caused by systemic effects of the epsilon toxin produced by the anaerobic bacterium Clostridium perfringens type D. The only practical means of controlling the occurrence of enterotoxaemia is to immunise animals by vaccination. The vaccine is prepared by deriving a toxoid from the bacterial culture filtrate and the potency of the vaccine is tested with the in vivo mouse neutralisation test (MNT). Due to ethical, economic and technical reasons, alternative in vitro assays are needed. In this study an indirect cytometric bead immunoassay (I-CBA) was developed for use in vaccine potency testing and the results were compared with those obtained using an indirect enzyme-linked immunosorbent assay (I-ELISA) and the MNT. Sera were collected from guinea pigs immunised with three different production batches of enterotoxaemia vaccine and the levels of anti-epsilon toxin antibodies were determined. Although the intra- and inter-assay variability was satisfactory, epsilon antitoxin levels determined by both the I-ELISA and indirect cytometric bead immunoassay (I-CBA) tests were higher than those of the MNT assay. In contrast to the MNT, all of the serum samples were identified as having antitoxin levels above the required minimum (not less than 5 U/mL). These results indicate that the respective in vitro tests in their current formats are not yet suitable alternatives to the in vivo MNT. The growing demand for a more humane, cost-effective and efficient method for testing the potency of enterotoxaemia vaccines, however, provides a strong impetus for further optimisation and standardisation of the I-CBA assay but further analytical research is required.

Quantitative anti-PA IgG ELISA; assessment and comparability with the anthrax toxin neutralization assay in goats.

BACKGROUND: Presently, few data exist on the level and duration of anti-protective antigen (PA) IgG in vaccinated livestock. Various adaptation of enzyme-linked immunosorbent assays (ELISAs) have been developed in studies to assess immune response following vaccination, albeit mostly in laboratory rodent models. The quantitative anti-anthrax IgG ELISA in this study describes a method of enumerating the concentration of anti-PA specific IgG present in sera of immunized goats, with the aid of an affinity-purified caprine polyclonal anti-anthrax PA-83 IgG standard. This was compared with the anthrax toxin neutralization assay (TNA) which measures a functional subset of toxin neutralizing anti-PA IgG. RESULTS: The measured concentrations obtained in the standard curve correlated with the known concentration at each dilution. Percentage recovery of the standard concentrations ranged from 89 to 98% (lower and upper asymptote respectively). Mean correlation coefficient (r2) of the standard curve was 0.998. Evaluation of the intra-assay coefficient of variation showed ranges of 0.23-16.90% and 0.40-12.46% for days 28 and 140 sera samples respectively, following vaccination. The mean inter-assay coefficient of variation for triplicate samples repeated on 5 different days was 18.53 and 12.17% for days 28 and 140 sera samples respectively. Spearman's rank correlation of log-transformed IgG concentrations and TNA titres showed strong positive correlation (rs = 0.942; p = 0.01). CONCLUSION: This study provides evidence that an indirect ELISA can be used for the quantification of anti-anthrax PA IgG in goats with the added advantage of using single dilutions to save time and resources. The use of such related immunoassays can serve as potential adjuncts to potency tests for Sterne and other vaccine types under development in ruminant species. This is the first report on the correlation of polyclonal anti-anthrax PA83 antibody with the TNA in goats.