Study On The Characteristion Of Lekotoxin Of Pathogenic Fusobacterium Necrophorum

This study successfully isolated and purified the natural leukotoxin from the culture supernatant of the high pathogenic bovine Fusobacterium necrophorum(FN) 5 strains by Cross-flow filtration, and the biochemical and biological characteristics and its immunoprotection of the leukotoxin were studied in vitro. Contents are as follows:The subspecies of FN5 was identified as subsp. necrophorum by PCR, the logarithmic phase of FN5 was determined by using the colony counting.The leukotoxicity of the cell-free culture supernatant of FN5 on bovine polymorphonuclear neutrophils leukocytes (PMNs) was determined by WST dye-reduction assay. The effect of store temperature and growth phase of bacterium on the leukotoxicity of culture supernatant was also examined.The crude leukotoxin was collected by cross-flow filtration apparatus ( Sartorious Stedium Biotech) at 4℃. SDS-PAGE indicated that the leukotoxin is highly unstable, for it degradated into several high-molecular-weigh polypeptides; and these proteins can recognized by the antiserum raised against recombinant FN4 leukotoxin truncated fragment BSBSE in western blot.The toxicity and the immunoprotection of crude leukotoxin were examined. It can cause ribbits to death, and also can protect host from the formation of liver abscesses even with experimental challenage with viable bacteria.Crude leukotoxin was polished by ultrafiltration, which retain the proteins that molecule weight greater than 100 kDa. The difference of pathogencity and immunoprotection between the crude leukotoxin and the purified leukotoxin were tested by animal experiment and transmission electron microscope (TEM), recpectively. The result revealed that the pathogencity of the polished leukotoxin was lower than crude leukotoxin, but the immunoprotection was similar.The pathological changes of the bovine PMNs treated with crude leukotoxin and polished leukotoxin, respectively, show the characteristics that consist with cells undergoing apoptosis, and the leukotoxicity of polished leukotoxin was lower than crude leukotoxin.The results from this study are suggestive of the immunogenicity and protective effects in rabbit and mice of the culture supernatant of FN5 containing leukotoxin, and this data can provided the foundation of the theory to prepare for a new leukotoxin subunit vaccine to immunoprophylaxis the liver abscesses and foot rot formation of cattle...