Immunoprophylaxis and molecular epidemiology of anthrax and the fate of Bacillus anthracis in living vectors and the environment of Namibia and South Africa
Final Report Abstract
The projects encompassed investigations of the genetic diversity and fate of B. anthracis circulating in livestock, wildlife and in environmental habitats in Namibia and South Africa. The roles of perennial and seasonal water holes and of living vectors like flies, amoeba and others were investigated. Another main part comprised studies in laboratory rodents and goats to compare the immunogenicity, protective efficacy and safety of recombinant peptide and DNA vaccines with those of the live spore vaccine (LSV) 34F2 (Sterne). Underpinning the science was the aim to support the academic and professional careers of young Namibian, South African and German scientists within the framework of a multi-national academic network. A newly established 31-marker MLVA together with analysis of SNPs and SNRs was used for epidemiological investigations. The MLVA database established comprises nearly 1200 isolates of B. anthracis, representing about 190 MLVA-genotypes. Data from the Namibian database became part of the MLVA database under http://mlva.u-psud.fr/mlvav4/genotyping/view.php. Efforts were successfully undertaken to harmonize a common MLVA coding system between German, Italian and South African institutions. During the project >500 environmental samples were collected over a period of four seasons and tested to determine the ratio of spores to vegetative bacilli and the total number of B. anthracis. The results revealed nil to very low numbers of spores in perennial water holes and low numbers of spores in seasonal water holes (gravel pits). The concentrations determined via semi-quantitative PCR argue against a meaningful role of such sites as infectious sources in the epidemiology of anthrax under the conditions of a highly endemic region. Three studies examined the fate of B. anthracis in either artificially infected or naturally occurring flies in the ENP in Namibia. From the results of these experiments the conclusion was drawn that a time dependent spread of vegetative cells or spores, taken up by a fly from a carcass and deposited on the vegetation, leads to variable viability of B. anthracis in the environment. Whether and to what extend they survive as infectious spores would then depend on their physiological status during deposition and, in case of vegetative cells being deposited, on the environmental conditions allowing or preventing sporulation. In any case, the survival in the gut of flies is very limited. For comparative studies on the immunogenicity, protective efficacy and safety of the LSV versus nonliving vaccine (NLV) candidates recombinant peptide vaccines based on components of the toxins, spore, and capsule in combination with a lipopeptide adjuvant, with and without formalin-inactivated spores (FIS), were tested in various combinations in mice, rabbits and finally goats. Moreover, appropriate DNA-vaccines designed for directed presentation of expressed antigens were tested in combination with vectors coding for regulatory adjuvants. In field experiments with goats the protein based NLVs showed up to 80% protection from a lethal challenge if FIS was part of the vaccine. This was equivalent to the LSV (60 – 100%). In contrast, the DNA-vaccines showed little to no immunogenicity in goats, though 90% protection from a lethal challenge was seen in mice. Surprisingly, our results indicate that antibodies generated against FIS in goats are not comprised of antibodies against BclA and that BclA seems to be poorly or not immunogenic in goats, in strong contrast to the mouse model. Recent publications have shown that antigens, physiologically located beneath the exosporium, can contribute to a protective immune response and should be considered for further veterinary vaccine tests. Immunological data from the study with the LSV indicate that the current veterinary immunization protocol, one dose with annual boosters, is suboptimal at best and can be improved. A passive mouse protection assay based on testing the protective capacity of animal sera in A/J mice challenged with the LSV proved to be reliable as a correlate for protection in the target animal.
Publications
- (2009): Review – Anthrax in animals. JMAM, 30, p. 481-489, ISSN: 0098- 2997
Beyer, W., P.C.B. Turnbull
- (2012): Distribution and Molecular Evolution of Bacillus anthracis Genotypes in Namibia. PLoS Negl Trop Dis 6(3): e1534
Beyer W., Bellan S., Eberle G., Ganz H.H., Getz W.M., Haumacher R, Hilss K.A., Kilian W., Lazak J., Turner W.C., Turnbull P.C.B.
(See online at https://doi.org/10.1371/journal.pntd.0001534) - (2012): Synthesis and immunochemical evaluation of a non-methylated disaccharide analogue of the anthrax tetrasaccharide. Org. Biomol. Chem., 10, 8524
Milhomme, O., Koehler, S.M., Ropartz, D., Lesur, D., Pilard, S., Djedaïni-Pilard, F., Beyer, W., Grandjean, C.
(See online at https://doi.org/10.1039/c2ob26131f) - (2013): A simple, reliable M'Fadyean stain for visualizing the Bacillus anthracis capsule. J. Microbio. Meth., 92: 264- 269
Owen, M.P., Schauwers, W., Hugh-Jones, M.E., Kiernan, J.A., Turnbull P.C.B., Beyer, W.
(See online at https://doi.org/10.1016/j.mimet.2013.01.009) - (2013): Co-infection of an animal with more than one genotype can occur in anthrax. Letters in Applied Microbiology 57:380-4
Beyer, W. and Turnbull, P.C.B.
(See online at https://doi.org/10.1111/lam.12140) - (2013): Effects of experimental exclusion of scavengers from carcasses of anthrax-infected herbivores on Bacillus anthracis sporulation, survival, and distribution. Appl. Environ. Microbiol., 79(12):3756
Bellan, S.E., Turnbull, P.C.B., Beyer, W., Getz, W. M.
(See online at https://doi.org/10.1128/AEM.00181-13) - (2014): Failure of Sterne- and Pasteur-Like Strains of Bacillus anthracis to Replicate and Survive in the Urban Bluebottle Blow Fly Calliphora vicina under Laboratory Conditions. PLoS ONE 9(1): e83860
von Terzi B., Turnbull P.C.B, Bellan S.E., Beyer W.
(See online at https://doi.org/10.1371/journal.pone.0083860) - (2014): Novel Giant Siphovirus from Bacillus anthracis Features Unusual Genome Characteristics. PLoS ONE 9(1): e85972
Ganz H.H., Law C., Schmuki M., Eichenseher F., Calendar R., Loessner M.J., Wayne M.G., Korlach J., Beyer W., Klumpp J.
(See online at https://doi.org/10.1371/journal.pone.0085972) - (2014): Quantitative anti-PA IgG ELISA; assessment and comparability with the anthrax toxin neutralization assay in goats. BMC Veterinary Research 2013, 9:265
Ndumnego, O.C., Crafford, J., Beyer W., van Heerden H.
(See online at https://doi.org/10.1186/1746-6148-9-265) - (2014): The odd one out: Bacillus ACT bacteriophage CP-51exhibits unusual properties compared to related Spounavirinae W.Ph.and Bastille. Virology 462-463, 299–308
Klumpp J., Schmuki M., Shanmuga S., Beyer W., Fouts D.E., Bernbach V., Calendar R., Loessner M.J.
(See online at https://doi.org/10.1016/j.virol.2014.06.012) - (2015): BclA and toxin antigens augment each other to protect NMRI mice from lethal Bacillus anthracis challenge. Vaccine
Volume 33, Issue 24, 4 June 2015, Pages 2771-2777
Koehler SM, Baillie LW, Beyer W
(See online at https://doi.org/10.1016/j.vaccine.2015.04.049)
