Vaccine potential of attenuated mutants of C. pseudotuberculosis in sheep

CRC for Vaccine Technology and Department of Microbiology and Immunology, The University of Melbourne, Parkville, Victoria, Australia.
Infection and Immunity (Impact Factor: 3.73). 03/1998; 66(2):474-9.
Source: PubMed


Corynebacterium pseudotuberculosis, a gram-positive facultative intracellular bacterial pathogen, is the etiological agent of the economically important disease caseous lymphadenitis (CLA) in both sheep and goats. Attenuated mutants of C. pseudotuberculosis have the potential to act as novel vaccines against CLA and as veterinary vaccine vectors. In this report, we have assessed the virulence of both aroQ and pld mutants of C. pseudotuberculosis in sheep and concurrently their capacity to act as vaccines against homologous challenge. The results suggest that aroQ mutants of C. pseudotuberculosis are attenuated with regard to both lymph node persistence and vaccination site reactogenicity. Immunologically, aroQ mutants failed to elicit detectable specific gamma interferon (IFN-gamma)-secreting lymphocytes and induced low levels of antibodies to C. pseudotuberculosis culture supernatant antigens. Following subcutaneous vaccination, the immune responses induced by aroQ mutants did not protect sheep from infection with the wild-type strain but did appear to reduce the clinical severity of disease resulting from challenge. Conversely, an attenuated C. pseudotuberculosis strain expressing an enzymatically inactive phospholipase D exotoxin, when used as a vaccine, elicited a protective immune response. Protection appeared to correlate with in vivo persistence of the vaccine strain, the induction of IFN-gamma-secreting lymphocytes, and relatively high levels of antibodies to culture supernatant antigens. The results suggest that aroQ mutants of C. pseudotuberculosis may be overly attenuated for use as a CLA vaccines or as vaccine vectors.

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Available from: Richard Strugnell, Apr 28, 2014
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    • "Three Boscat rabbits weighing 2.5 Kg were inoculated subcutaneously with 1 ml dose containing 25 μg rPLD protein mixed with complete Freund's adjuvant (Sigma) followed by 2 doses of 25 μg rPLD mixed with incomplete Freund's adjuvant (Sigma) at weekly intervals. One week post last dose antibodies were assayed by ELISA (23) using rPLD protein as a coating antigen. Titers were expressed as the reciprocal of the dilution which gave an OD three fold above the OD of preimmune serum analysed on the same plate. "
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    ABSTRACT: Fourteen isolates of Corynebacteruim pseudotuberculosis of them 7 were isolated from sheep with Caseous Lymphadenitis "biotype 1" and 7 isolated from buffaloes with Oedematous Skin Disease "biotype 2". All isolates were identified by standard microbiological techniques and by polymerase chain reaction targeting, 16S rRNA and phospholipase D genes. Synergistic haemolytic titers of all isolates were assayed by plate technique. The presences of phospholipase D gene in supernatants of all isolates were performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis immunoblot technique by using hyperimmune serum raised in rabbit immunized with recombinant phospholipase D gene antigen. The concentration of phospholipase D gene was assayed by scanning the bound phospholipase D gene with specific antibodies that appeared at 31.5 kDa. Results presented that there is no correlation between titer of Synergistic haemolytic activity and the actual phospholipase D genes concentration in culture supernatants. Also results presented that Synergistic haemolytic activity and phospholipase D genes produced by biotype 2 (buffalo isolates) was generally higher than those by biotype 1(sheep isolates).
    Full-text · Article · Apr 2012 · Brazilian Journal of Microbiology
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    • "The strains 1002 and C231 of Corynebacterium pseudotuberculosis were used in this study. Strain 1002 was isolated from an infected goat in Brazil and has been shown to be naturally low virulent [23,56]; strain C231 was isolated from an infected sheep in Australia, and it showed a more virulent phenotype [24]. Species confirmation was performed by biochemical and molecular methods for both strains, as described [77]. "
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    ABSTRACT: Background Bacterial exported proteins represent key components of the host-pathogen interplay. Hence, we sought to implement a combined approach for characterizing the entire exoproteome of the pathogenic bacterium Corynebacterium pseudotuberculosis, the etiological agent of caseous lymphadenitis (CLA) in sheep and goats. Results An optimized protocol of three-phase partitioning (TPP) was used to obtain the C. pseudotuberculosis exoproteins, and a newly introduced method of data-independent MS acquisition (LC-MSE) was employed for protein identification and label-free quantification. Additionally, the recently developed tool SurfG+ was used for in silico prediction of sub-cellular localization of the identified proteins. In total, 93 different extracellular proteins of C. pseudotuberculosis were identified with high confidence by this strategy; 44 proteins were commonly identified in two different strains, isolated from distinct hosts, then composing a core C. pseudotuberculosis exoproteome. Analysis with the SurfG+ tool showed that more than 75% (70/93) of the identified proteins could be predicted as containing signals for active exportation. Moreover, evidence could be found for probable non-classical export of most of the remaining proteins. Conclusions Comparative analyses of the exoproteomes of two C. pseudotuberculosis strains, in addition to comparison with other experimentally determined corynebacterial exoproteomes, were helpful to gain novel insights into the contribution of the exported proteins in the virulence of this bacterium. The results presented here compose the most comprehensive coverage of the exoproteome of a corynebacterial species so far.
    Full-text · Article · Jan 2011 · BMC Microbiology
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    • "Thus, Simmons and colleagues (1997) obtained C. pseudotuberculosis strains that lack virulence through attenuation of aroB and aroQ gene, using allelic exchange. Such strains were unable to cause CLA in murine models, suggesting its use as vaccine vectors and a potential attenuated vaccine in the control of CLA (Simmons et al., 1998). "
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    ABSTRACT: Corynebacterium pseudotuberculosis, a Gram-positive intracellular pathogen, is the etiological agent of caseous lymphadenitis or CLA. This bacterium infects goats and sheep and causes great economic losses worldwide annually, mainly for goat producers. Despite its importance, CLA is still poorly characterized. However, with advances in the genomic field, many C. pseudotuberculosis genes have already been characterized, mainly those related to virulence such as phospholipase D. Here, we examined the use of the several available genes of C. pseudotuberculosis and reviewed their applications in vaccine construction, more efficient diagnostics for CLA, and control of this disease, among other applications.
    Full-text · Article · Feb 2008 · Genetics and molecular research: GMR
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