Pasteurella sp. associated with fatal septicaemia in six African elephants.

The sudden mortality of African elephants (Loxodonta africana) in Botswana and Zimbabwe in 2020 provoked considerable public interest and speculation. Poaching and malicious poisoning were excluded early on in the investigation. Other potential causes included environmental intoxication, infectious diseases, and increased habitat stress due to ongoing drought. Here we show evidence of the mortalities in Zimbabwe as fatal septicaemia associated with Bisgaard taxon 45, an unnamed close relative of Pasteurella multocida. We analyse elephant carcasses and environmental samples, and fail to find evidence of cyanobacterial or other intoxication. Post-mortem and histological findings suggest a bacterial septicaemia similar to haemorrhagic septicaemia caused by P. multocida. Biochemical tests and 16S rDNA analysis of six samples and genomic analysis of one sample confirm the presence of Bisgaard taxon 45. The genome sequence contains many of the canonical P. multocida virulence factors associated with a range of human and animal diseases, including the pmHAS gene for hyaluronidase associated with bovine haemorrhagic septicaemia. Our results demonstrate that Bisgaard taxon 45 is associated with a generalised, lethal infection and that African elephants are susceptible to opportunistically pathogenic Pasteurella species. This represents an important conservation concern for elephants in the largest remaining metapopulation of this endangered species.

Immunogenicity and Protective Efficacy of a Non-Living Anthrax Vaccine versus a Live Spore Vaccine with Simultaneous Penicillin-G Treatment in Cattle.

: Sterne live spore vaccine (SLSV) is the current veterinary anthrax vaccine of choice. Unlike the non-living anthrax vaccine (NLAV) prototype, SLSV is incompatible with concurrent antibiotics use in an anthrax outbreak scenario. The NLAV candidates used in this study include a crude recombinant protective antigen (CrPA) and a purified recombinant protective antigen (PrPA) complemented by formalin-inactivated spores and Emulsigen-D®/Alhydrogel® adjuvants. Cattle were vaccinated twice (week 0 and 3) with NLAVs plus penicillin-G (Pen-G) treatment and compared to cattle vaccinated twice with SLSV alone and with Pen-G treatment. The immunogenicity was assessed using ELISA against rPA and FIS, toxin neutralisation assay (TNA) and opsonophagocytic assay. The protection was evaluated using an in vivo passive immunisation mouse model. The anti-rPA IgG titres for NLAVs plus Pen-G and SLSV without Pen-G treatment showed a significant increase, whereas the titres for SLSV plus Pen-G were insignificant compared to pre-vaccination values. A similar trend was measured for IgM, IgG1, and IgG2 and TNA titres (NT50) showed similar trends to anti-rPA titres across all vaccine groups. The anti-FIS IgG and IgM titres increased significantly for all vaccination groups at week 3 and 5 when compared to week 0. The spore opsonising capacity increased significantly in the NLAV vaccinated groups including Pen-G treatment and the SLSV without Pen-G but much less in the SLSV group with Pen-G treatment. Passive immunization of A/J mice challenged with a lethal dose of 34F2 spores indicated significant protective capacity of antibodies raised in the SLSV and the PrPA + FIS + adjuvants vaccinated and Pen-G treated groups but not for the NLAV with the CrPA + FIS + adjuvants and the SLSV vaccinated and Pen-G treated group. Our findings indicate that the PrPA + FIS + Emulsigen-D®/Alhydrogel® vaccine candidate may provide the same level of antibody responses and protective capacity as the SLSV. Advantageously, it can be used concurrently with Penicillin-G in an outbreak situation and as prophylactic treatment in feedlots and valuable breeding stocks.

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 Non-Living Anthrax Vaccine Candidates in Cattle and Protective Efficacy of Immune Sera in A/J Mouse Model Compared to the Sterne Live Spore Vaccine.

The Sterne live spore vaccine (SLSV, Bacillus anthracis strain 34F2) is the veterinary vaccine of choice against anthrax though contra-indicated for use with antimicrobials. However, the use of non-living anthrax vaccine (NLAV) candidates can overcome the SLSV limitation. In this study, cattle were vaccinated with either of the NLAV (purified recombinant PA (PrPA) or crude rPA (CrPA) and formaldehyde-inactivated spores (FIS of B. anthracis strain 34F2) and emulsigen-D®/alhydrogel® adjuvants) or SLSV. The immunogenicity of the NLAV and SLSV was assessed and the protective efficacies evaluated using a passive immunization mouse model. Polyclonal IgG (including the IgG1 subset) and IgM responses increased significantly across all vaccination groups after the first vaccination. Individual IgG subsets titres peaked significantly with all vaccines used after the second vaccination at week 5 and remained significant at week 12 when compared to week 0. The toxin neutralization (TNA) titres of the NLAV vaccinated cattle groups showed similar trends to those observed with the ELISA titres, except that the former were lower, but still significant, when compared to week 0. The opsonophagocytic assay indicated good antibody opsonizing responses with 75% (PrPA+FIS), 66% (CrPA+FIS) and 80% (SLSV) phagocytosis following spores opsonization. In the passive protection test, A/J mice transfused with purified IgG from cattle vaccinated with PrPA+FIS+Emulsigen-D®/Alhydrogel® and SLSV had 73% and 75% protection from challenge with B. anthracis strain 34F2 spores, respectively, whereas IgG from cattle vaccinated with CrPA+FIS+Emulsigen-D®/Alhydrogel® offered insignificant protection of 20%. There was no difference in protective immune response in cattle vaccinated twice with either the PrPA+FIS or SLSV. Moreover, PrPA+FIS did not show any residual side effects in vaccinated cattle. These results suggest that the immunogenicity and protective efficacy induced by the NLAV (PrPA+FIS) in the cattle and passive mouse protection test, respectively, are comparable to that induced by the standard SLSV.

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.