Abstract
Phospholipase D (PLD) is the major virulence factor in Corynebacteruim pseudotuberculosis that exhibit synergistic haemolysis (SH) of sheep blood cells in the presence of products from Rhodococcus equi. To evaluate the correlation between SH activity and the actual concentration of PLD involved in culture supernatants of C. pseudotuberculosis obtained from sheep with Caseous Lymphadenitis (CLA) and buffaloes with Oedematous Skin Disease (OSD). Fourteen isolates of C. pseudotuberculosis isolated from sheep with CLA and buffaloes with OSD “biotype 2” were identified by standard microbiological techniques and by multiplex PCR assay for direct detection of 16S rRNA gene, rpoB gene and pld gene specific for identification of C. pseudotuberculosis isolates. SH titers of all isolates were assayed by plate technique. The presences of PLD gene in supernatants of all isolates were performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis immunoblot technique. The concentration ofPLD gene was assayed by scanning the bound PLD gene with specific antibodies that appeared at 31.5 kDa. Results presented that multiplex PCR is rapid and specific for detection of C. pseudotuberculosis isolates and there is no correlation between titer of SH activity and the actual PLD genes concentration in culture supernatants. Moreover, the SH activity of PLD genes produced by biotype 2 was generally higher than those by biotype 1. Key words: Corynebacteruim pseudotuberculosis, OSD, CLA, PLD, multiplex PCR, synergistic haemolysis.
Highlights
Phospholipase D (PLD) is a potent exotoxin produced by Corynebacteruim pseudotuberculosis of sheep origin (Hodgson et al, 1990; Lipsky et al, 1982; Tashjian and Campbell, 1995) and by buffalo isolates (Ghoneim et al, 2001)
Fourteen isolates of C. pseudotuberculosis isolated from sheep with Caseous Lymphadenitis (CLA) and buffaloes with Oedematous Skin Disease (OSD) “biotype 2” were identified by standard microbiological techniques and by multiplex PCR assay for direct detection of 16S rRNA gene, rpoB gene and pld gene specific for identification of C. pseudotuberculosis isolates
Multiplex PCR targeting three C. pseudotuberculosis genes: (a) the 16S rRNA gene, which is the gene of choice for most microbial taxonomy studies (Çetinkaya et al, 2002; Khamis et al, 2005); (b) the RNA polymerase ß-subunit gene, which is currently used for the study of phylogenetic relationships in the genera Corynebacterium and Mycobacterium (Dorella et al, 2006); and (c) genes encoding exotoxin PLD, which is a sphingomyelinase implicated in the virulence of C. pseudotuberculosis, C. ulcerans and Arcanobacterium haemolyticum (McNamara et al, 1995)
Summary
Phospholipase D (PLD) is a potent exotoxin produced by Corynebacteruim pseudotuberculosis of sheep origin (Hodgson et al, 1990; Lipsky et al, 1982; Tashjian and Campbell, 1995) and by buffalo isolates (Ghoneim et al, 2001). Depending upon the information that PLD is the major virulence factor in C. pseudotuberculosis, many significant efforts have been made to produce effective caseous lymphadenitis (CLA) vaccines. The majority of prepared vaccines were derived from PLD-rich culture supernatants, inactivated with formalin to produce toxoid vaccine (Egen et al, 1989; Paton et al, 2003; Piontkowski et al, 1998; Williamson, 2001). Other researchers reported that toxoid vaccines and inactivated corynebacterial cells (bacterins) provide partial protection (Brogden et al, 1996)
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