الفهرس 
IntroductionOnly 14 pages are availabe for public view
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Abstract
To study these genetic functions regulating biofilm formation and EPS production by H.somni, mutants of H.somni were generated using the EZ::Tn5TM <KAN-2>Tnp Transposome and screened for altered biofilm formation. Twenty-five mutants were identified that produced 50-70% less biofilm than the parent strain, which was confirmed by scanning electron microscopy. These mutants are genetically stable as determined by southern blot hybridization. The transposon insertion sites of these biofilm defective mutants were identified by sequencing of inverse PCR products. Some biofilm deficient mutants had the transposon located in different regions of a homolog of filamentous haemagglutinin (FHA), predicted to be involved in attachment. Other mutants had mutations in a gene encoding for an outer membrane efflux protein (TolC), which aids in intracellular trafficking and secretion and predicted to aid in antibiotic resistance. Some of the FHA mutants that formed a diminished biofilm also produced less exopolysaccharide (EPS), which was determined by alcian blue silver staining, ELISA and the amount of EPS that could be isolated. These mutants were FHA mutants which might explain the role played by EPS in the attachment of biofilm to surfaces. The exopolysaccharide was isolated in greatest quantity from the sedimentary cellular mass present in broth cultures grown under low oxygen tension for 5 days, indicating that it was upregulated under stress conditions and during biofilm formation. Moreover, maximum amounts of EPS were produced during stationary phase, under anaerobic growth conditions, and during growth in 2% NaCl which are stress conditions that favors biofilm formation. That means that maximum amount of EPS can be obtained under growth conditions that stimulated biofilm formation and that is a component of the biofilm matrix. Some sugars (Mannitol, glucose, and mannose) appeared to stimulate EPS biosynthesis. Furthermore, a preliminary investigation using ELISA for testing sera from calves prior to and after challenge with H.somni showed a modest rise in antibody titers to the EPS, indicating that the EPS may have potential as an antigen for serological diagnosis of H. somni infection. PCR using species specific primers based on the 16S rDNA gene allowed direct identification of H.somni infected cattle from lung samples at necropsy where two samples showed positive amplification out of 15 lung samples collected at necropsy from cattle suffering from pneumonia symptoms.