Development And Application Of Fusobacterium Necrophorum Subunit Vaccine

Fusobacterium necrophorum is a strictly anaerobic gram-negative polymorphic bacillus.Its pathogenicity factors include endotoxin,leukocyte toxin,platelet agglutination factor,hemagglutinin and hemolysin and so on,among which the main pathogenic factor is leukotoxin.The disease caused by Fusobacterium necrophorum occurred in different countries.When ruminants such as deer,sheep and other ruminants are infected with Fusobacterium necrophorum,they show claudication,swelling in joints,Liver abscess and“foot rot disease”,which caused great economic losses to farm.In this study we collected hoof samples from deer in a deer farm in Hubei Province to isolate Fusobacterium necrophorum and then prepare for the bacterial subunit vaccine which intends to provide some reference for clinical treatment and prevention of ruminant diseases caused by the bacteria.1.Isolation and identification of Fusobacterium necrophorumThe present study was performed to find the pathogen of rotted hoof sika deer?NO:052?at the farm located in Enshi state,Hubei province China.The anaerobe was isolated from rotted and healthy hoof samples of sika deer which were collected and cultured in BHI medium.Isolate was identified as Fusobacterium funduliforme by morphologic observation,biochemical tests,16S rRNA sequencing,bacterial biochemical identification,susceptibility tests and virulence tests.The bacteria were gram-negative bacilli,filaments of varying lengths under microscope.The strain showed 99%homology with Fusobacterium funduliforme through 16S rRNA gene sequencing,named HNFnf.It was concluded that it belonged to Fusobacterium necrophorum faculty by the determination of Vitek Bio-chemical identification instrument.Bacterium was intermediate susceptible to penicillin,cephalosporins and completely resistant to amikacin,and gentamycin from the susceptibility to antimicrobials tests.The study provided reference for clinical treatment of“foot rot disease”and“liver abscess”infected with Fusobacterium necrophorum in ruminal animals.2.Establishment of liver abscess model in rabbitsIn this research,Fusobacterium necrophorum HNFnf isolates were injected into rabbits by the method of rabbit ear vein injection with different gradient cfu.Fusobacterium necrophorum infection model had been established in rabbits by observing the clinical pathologic changes?anatomical pathological changes and histopathological changes of rabbits.The minimum lethal dose of the bacterium was 1.2*10¹⁴ CFU with which all rabbits died in 7 days.Before death rabbit performed like,food and water intake reducing,weight loss and so on.From the anatomical results of the dead rabbits,they showed lung surface bleeding;uniform gray and yellow liver abscess formation;spleen swelling;flatulence in intestinal tracts;urinary bladder accumulation.Histopathological results indicated that Aizen liver abscess formed with inflammatory cells infiltrated,innate structure of hepatic lobule disappeared,fatty degeneration occurred in liver cells;pink cellulose exuded in alveoli,which provided criteria for vaccine evaluation.3.The development and assessment for subunit vaccine of Fusobacterium necrophorumIn this study,bacterial intracellular proteins were obtained by lysis of bacteria and used as immunogenic substances to prepare for the vaccine which was developed according to the rules of Animal Biological products.Meanwhile,we also prepared killed bacterin,and then evaluation and comparison of vaccines were based on cost of preparation and prevention rates.The vaccine evaluation was assessed referring the rabbit liver abscess model described in the research two.The preparation of the two vaccines provides some certain guarantee for the clinical prevention of ruminant diseases caused by the bacteria.At the same time,it also provides some references for the development of the new vaccine.