Isolation and identification of Malaysian Pasteurella species responsible for small ruminant pneumonia for the purpose of developing an effective strain-specific vaccine

Abstract

The aims of this study were to identify the prevalance and distribution of Pasteurella haemolytica serotypes causing pneumonic pasteurellosis in sheep and goats in Malaysia, assess the efficacy of a novel P'. haemolytica vaccine in field trials and to isolate, characterise and assess the immunological significance of the polysaccharide capsule of P. haemolytica A2. The serotyping study indicated that there was little difference in the relative frequency of occurrence of A serotypes in the UK and in Malaysia. Serotype A2 was by far the most common and the other A serotypes did not differ significantly in order of prevalence. However, T biotypes appeared to be very rare in Malaysia compared to UK. When electrophoretic protein and lipopopolysaccharide profiles of some common strains from both countries were compared there appeared to be no significant differences among the strains. Four to five major protein bands with about twenty minor bands were shown to be present. The lipopolysaccharides profiles were of rough-type.A new P. luicnio/viiid iron-regulated protein (IRP) vaccine was tested in field studies for its efficacy in preventing pasleurellosis in Malaysian sheep farms. The results showed that this vaccine generated an immune response as measured in LLISA and IIIA tests. I he antibody liters were significantly higher in the vaccinated sheep and there appeared to be protection against clinical pasleurellosis following vaccination.The capsular polysaccharide antigen of P. haemolytica A2 was identified as an important immunogen and as a candidate for a future Pasteurella vaccine. An outer membrane protein-polysaccharide (OMP-PS) complex was successfully prepared from an ovine isolate of P. haemolytica serotype A2 by precipitation from log phase culture supernatant and subsequent purification by column chromatography. The optimum production of the complex was determined to be in 6 hour culture and comprised protein and polysaccharide (4.1 w/w) with low in lipopolysaccharide content.This complex was immunogenic in mice and adult sheep with induction of humoral anti capsular antibody in indirect heamagglulination (IHA) test and anti-OMP antibodies in immunoblolting. Direct immunisation of mice with the complex vaccine demonstrated significant protection against A2 infection. The sera from two-year old sheep immunised with OMP-PS passively protected mice against A2 challenge. Three month old lambs immunised with the same vaccine did not respond serologically and this sera did not passively protect mice.In vitro studies using ovine and murine macrophages indicated that the mechanism of protection is antibody mediated phagocytosis as the opsonophagocytic activity of immune sera could be demonstrated. These results indicate the potential of a /' . hitemolytica A2 OMP-PS complex vaccine to immunise adult sheep and suggests that passive protection of lambs against A2 infection is obtainable.