The Research Of Immune-enhancing Effects Of Robinia Pseudoacacia Polysaccharide On Mink Klebsiella Pneumoniae Inactivated Vaccine

For a long time, people regard Klebsiella pneumoniae(K. pneumoniae) as an opportunistic pathogen, with potential risk to the animal husbandry that has not been paid enough attention to. However, in recent years, it is reported that mink diseases caused by K. pneumoniae has a rapid increase, coupled with the widespread use of a variety of antimicrobial drugs, resulting in increased drug resistance and emergence of multi-drug-resistant K. pneumoniae strains.The damage that K. pneumoniae brings to mink breeding industry has became more and more serious. Thus with the changes of the environment, K. pneumoniae, as a pathogen of zoonotic diseases, should be widely concerned. Currently on the market there is still no effective K. pneumoniae bacteria vaccine for the prevention and treatment of this disease, resulting in no reasonable and effective prevention and treatment for K. pneumoniae, seriously restricting the development of the mink breeding industry. Therefore,in this study, Robinia Pseudoacacia Polysaccharide(RPPS), Freund’s adjuvant were selected as adjuvant to prepare K. pneumoniae inactivated vaccine, which provided novel technical support and theoretical basis to the prevention and control of K. pneumoniae. 1. Isolation and identification of K. pneumoniae isolated from mink in parts of ShandongThis study focused on the main bacterial pathogens which caused the death of mink in the last two years.The fresh samples were examined by clinical analysis and pathology observation on the basis of epidemiological investigation to identify the main pathogen. Meanwhile, pathological tissues in liver, lung and trachea were isolated to purify pathogenic bacteria. Then the bacteria were identified by drug susceptibility test, pathogenicity test, the morphological characteristics, physiological and biochemical characteristics were examined, in addition, 16 S rRNA gene sequencing were used for the molecular diagnosis. The results showed that the identical bacteria were examined from lung, liver and trachea, besides, the isolated strains were highly sensitive to ceftriaxone and sulbactam. From a comprehensive analysis, the isolated strains were K. pneumoniae. 2. Evaluation the effects of RPPS on the K. pneumoniae inactivated vaccineRPPS was prepared in our laboratory by the method of water extraction and ethanol precipitation. The K. pneumoniae inactivated with formaldehyde was mixed with RPPS or Freund’s adjuvant at a ratio of 1:1. A total of 160 mice(female) were randomly separated into four groups. Each mouse in groups I-IV was injected with the RPPS adjuvant K. pneumoniae inactivated vaccine, the Freund’s adjuvant K. pneumoniae inactivated vaccine, the K. pneumoniae inactivated vaccine, empty plasmid and PBS at 1, 7, and 14 days old, respectively. At 7, 14, 21, 28, 35, 42 and 49 days post the first inoculation, three different mice from each group were selected randomly to determine the serum antibody, IL-2 and IL-4 concentrations, and CD4+ and CD8+ T lymphocytes counts in peripheral blood. After the third inoculation, 20 mice in each group were challenged intraperitoneally with 10 LD50 K. pneumoniae strain. Survival status of mice was recorded for seven successive days after the challenge. The results showed that the K. pneumoniae inactivated vaccine obviously induced the production of antibody, the secretion of cytokines, the increase of CD4+ and CD8+ T lymphocytes counts in peripheral blood, and it provided a protection rate of 50% in K. pneumoniae challenged mice. Notably, mice inoculated with the RPPS adjuvant K. pneumoniae inactivated vaccine and Freund’s adjuvant K. pneumoniae inactivated vaccine had higher levels of immune responses than those inoculated with the pure K. pneumoniae inactivated vaccine, whereas RPPS has better immunologic enhancement as adjuvant than Freund’s adjuvant.