| Klebsiella pneumoniae(K. Pneumoniae) is an opportunistic pathogen which can cause a variety of animal diseases. It belongs to Enterobacter producing extended-spectrumβ-lactamases(ESBLs), which can hydrolyze broad spectrum cephalosporin antibiotics containing oxygen imino side chain and single amine antibiotics, and mediated bacteria producing resistance to these antibiotics. This bacteria majorly colonize in the respiratory tract and intestines of sick animals and human, leading to respiratory, digestive, urinary tract infections. The bacteria producing β- lactamase possess multiple antibiotics resistance property, making clinical treatment become a thorny issue. Over the years, K. Pneumoniae had not been taken seriously because that it was believed with little risk on livestock and poultry. However, in recent years, with the increasing reports of fur animal diseases caused by K. Pneumoniae and the property of antibiotic resistance, as the pathogen of zoonotic disease, K. Pneumoniae has aroused wide attention. As a protective antigen and virulence factor of encapsulated bacteria, capsular polysaccharide(CPS) is one of the most important target antigen and has the potential to be exploited to a subunit vaccine. Therefore, in this study, we extracted CPS of K. Pneumoniae and prepared it to subunit vaccines with Taishan Pinus massoniana pollen polysaccharides(TPPPS) and propolis used as adjuvants, which provided novel technical support and theoretical basis to the prevention and control of K.Pneumoniae.From the pathological tissues in liver, lung, trachea of fur animals, bacteria were isolated and purified, which were identified through observation of morphological characteristics, biochemical tests, susceptibility tests and 16 S rRNA gene sequencing detection. The results showed that the bacteria isolated from lung, liver, trachea tissues were identical. From a comprehensive analysis, the isolates were K. Pneumoniae.The K. Pneumoniae capsular polysaccharides(CPS-K) were extracted by the methods of cetyl trimethyl ammonium bromide(CTAB) precipitation polysaccharides, ethanol elimination nucleic acids and phenol elimination protein. Then subunit vaccines were prepared with CPS-K and TPPPS, propolis and Freund’s adjuvant mixed at the ratio of 1:1respectively. A total of 40 male Kunming mice were randomly divided into four groups(I-Ⅳ),which were vaccinated with TPPPS adjuvant CPS subunit vaccine, propolis adjuvant CPS subunit vaccine, Freund’s adjuvant CPS subunit vaccine, and CPS subunit vaccine at 3, 7, 14 days old respectively. At 7, 14, 21, 28, 35, 42, 49, 56 days post the first vaccination,T-lymphocyte transformation rate, serum antibody titers, serum IL-2 concentrations, CD4+and CD8+T lymphocytes counts in peripheral blood were detected.The results showed that CPS subunit vaccine could not stimulate produce enough protective antibodies, and TPPPS,propolis and Freund’s adjuvant could significantly enhance the levels of cellular and humoral immunity. Furthermore, the levels of cellular and humoral immunity in group I were significantly higher than those in group II and III(p < 0.05). Therefore, TPPPS has good immunologic enhancement as adjuvant. This study has provided theoretical foundation for developing K. Pneumoniae CPS subunit vaccine. |
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