Isolation Identification of Klebsiella Pneumoniae Isolated from Fur Animal and the Preparation of Klebsiella Pneumoniae Microcapsule Oral Vaccine
|Shandong Agricultural University
|Preventive Veterinary Medicine
|Klebsiella pneumoniae 23S rRNA microcapsule oral vaccine immunologicalfunction
For a long time, people regard Klebsiella pneumoniae as an opportunistic pathogen, withpotential risk to the animal husbandry that has not been paid enough attention to. However, inrecent years, it is reported that fur animal diseases caused by Klebsiella pneumoniae has arapid increase, coupled with the widespread use of a variety of antimicrobial drugs, resultingin increased drug resistance and emergence of multi-drug-resistant Klebsiella pneumoniaestrains.The damage that Klebsiella pneumoniae brings to fur animal breeding industry hasbecame more and more serious. Thus with the changes of the environment, Klebsiellapneumoniae, as a pathogen of zoonotic diseases, should be widely concerned.In this study, based on the traditional physical and chemical characterization12representative strains of animal origin Klebsiella pneumoniae,23S rRNA gene sequenceanalysis were carried out to construct a phylogenetic tree, the fungal species differences werecomparied and laid the foundation for systematic studies at the molecular level and geneticdiversity of Klebsiella pneumoniae, a rapid and simple identification of the Klebsiellapneumoniae and23S rRNA gene sequence analysis were established. Currently on the marketthere is still no effective Klebsiella pneumoniae bacteria vaccine for the prevention andtreatment of this disease, resulting in no reasonable and effective prevention and treatment forKlebsiella pneumoniae, seriously restricting the development of the fur animal breedingindustry. Therefore,in this study, Taishan pine pollen polysaccharide, propolis were selected asadjuvant, the microcapsules were prepared to make Klebsiella pneumoniae vaccinemicrocapsules, and oral immunization of mice were carried out to detect immune responses. Itis an important progress for Klebsiella pneumoniae prevention, at the same time laid thefoundation for a new oral vaccine and the development of controlling release formulations.1. Isolation identification and phylogenetic analysis of Klebsiellapneumoniae isolated from fur animal in parts of ShandongThis study focused on the main bacterial pathogens which caused the death of fur-bearinganimals （raccoon dog, fox, mink） in the last two years.The fresh samples were examined byclinical analysis and pathology observation on the basis of epidemiological investigation toidentify the main pathogen. Meanwhile, pathological tissues in liver, lung and trachea were isolated to purify pathogenic bacteria. Then the bacteria were identified by drug susceptibilitytest, pathogenicity test，the morphological characteristics, physiological and biochemicalcharacteristics were examined, in addition,16S rRNA gene sequencing were used for themolecular diagnosis. The results showed that the identical bacteria were examined from lung,liver and trachea, besides, the isolated strains not only were highly sensitive to ceftriaxoneand sulbactam, but also had strong pathogenicity to mice. What is more, they were alsoproved to have high similarity （98.6%-100%） with the16S rRNA gene of Klebsiellapneumoniae from GenBank database. From a comprehensive analysis, the isolated strainswere Klebsiella pneumoniae of Klebsiella generic.2. Sequence analysis of23S rRNA genes of different animal speciesKlebsiella pneumoniaeTo assess the evolution of Klebsiella pneumoniae by23S rRNA genes, the fragments（727bp） of23S rRNAgenes were amplified by polymerase chain reaction （PCR） fromfour mink Klebsiella pneumoniae strains, two raccoon dog strains, two fox Klebsiellapneumoniae strains, two porcine Klebsiella pneumoniae strains and two fowl Klebsiellapneumoniae strains. The PCR products were cloned and sequenced. The nucleotideshomology analysis of the23S rRNA gene was carried out using the BLAST. Multiplesequences alignments and the phylogenetic tree were performed using the DNAStar.software.The result showed that the homologies between twelve Klebsiella pneumoniae strains were96.8%-99.0%with the reported one strain in GenBank, were90.8%-91.7%%with Salmonellaand Proteus mirabilis strains; The homologies between eight fur animal Klebsiellapneumoniae strains were98.1%-99.9%, were95.5%-98.1%with porcine Klebsiellapneumoniae strains, and were96.7%-97.9%with fowl Klebsiella pneumoniae strains; Thehomologies between porcine Klebsiella pneumoniae strains and fowl Klebsiella pneumoniaestrains were95.9%-97.4%. The different animal species Klebsiella pneumoniae strains had acertain degree of genetic difference.23S rRNA sequence analyses may be a reliable and rapidway for identification of different animal species Klebsiella pneumoniae. 3. The preparation of Klebsiella pneumoniae microcapsule oralvaccine and its immunological functionThis study aims to explore the immunogenic effects of an oral Klebsiella pneumoniaemicrocapsule vaccine. The oral K. pneumoniae microcapsule vaccine was prepared through amodified spray drying and boundary coordination method. Up to210healthy unvaccinatedKunming mice (male) were randomly assigned into five groups: a Taishan Pinus massonianapollen polysaccharide microcapsule oral vaccine group, a propolis microcapsule oral vaccinegroup, a bacteria microcapsule vaccine group, a bacteria vaccine group and a blank controlgroup. The mice were orally vaccinated. On days3,7,14,21,28,35,42and49after the firstvaccination, the antibody titers, interleukin-2levels (IL-2), T-lymphocyte proliferation rates,the percentage of CD4+and CD8+T cells in the peripheral blood and the duodenal secretedIgA levels (SIgA) were measured. The average diameter of the microcapsule oral vaccineswas6.980μm. The microcapsule oral vaccine was soluble in intestinal juice and resisted thehydrochloric acid in gastric juice. The immune index of the Taishan Pinus massoniana pollenpolysaccharide microcapsule oral vaccine group was significantly higher than these of theother groups. Therefore the microcapsule oral vaccine prepared in this study activates cellularhumoral and local mucosal immunity as well as stimulates the production of SIgA and IgG.This method is easy to apply, safe and effective.