Isolation And Identification Of Mink Pseudomonas Aeruginosa And Study Of The Influence Of Inactivated Vaccine On Production Performance Of Mink

Mink hemorrhagic pneumonia is caused by Pseudomonas aeruginosa andmainly occurs in summer and autumn. This kind of disease is acute and fatal for mink.Mink for all ages is highly sensitive to this disease which has a high morbidity andlethality. It has become one of the most serious diseases like mink parvoviral enteritisand mink distemper which are the huge obstacles for the mink farming industry.This study isolated some P. aeruginosa strains from dead minks which weresuspected of being infected with hemorrhagic pneumonia in Shandong and Liaoningin2012. We identified22P. aeruginosa strains according to the growth phenotypes,biochemical characterizations and characteristics of the molecular biology of thebacteria. Based on the random amplified polymorphic of DNA and antimicrobialsusceptibility, we analysed the genetic relationship between the strains andsusceptibility rate to the antibiotics used often in the clinical application. And this willbe a good foundation for prevention and control of mink hemorrhagic pneumonia.The outer membrane protein F(OprF) of P. aeruginosa is highly conservedamong the different serotype strains and thought to be protective antigen. Thus, weamplified the gene of OprF from the strains had been identified correctly by PCR andcloned this gene into pMD18-T vector, sequenced and analysed. The right clone ofrecombinant vector was connected in pET-28a and transformed into the E. coliBL21.The protein OprF was obtained and identified by IPTG induction, ultrasoundlysis, purification, SDS-PAGE and Western-blot.Based on the protein OprF as coating antigen, a series of tests were carried outto screen the optimal situation to establish the indirect ELISA. The standardization ofindirect ELISAis with the concentration of OprF2μg/mL and positive serum1:4. The established ELISA’s cut-off value was0.06; limit of detection was1:256.Thecoefficient of variation of Intro-batch was0.33%～8.27%as well as the Inter-batchwas1.24%～5.59%. The established ELISA showed great specificity because thismethod could distinguished anti-serum against P. aeruginosa from other anti-serumsagainst some common pathogens of mink like Salmonella typhimurium,Staphylococcus aureus, Klebsiella pneumonia and Escherichia coli. The agreementrate of the established ELISA was93.3%when30known positive serum sampleswere tested by this method. This result means it can be an effective tool to evaluatethe antibody titer of P. aeruginosa and the infected situation of this disease in minkherd.This part we made the inactivated vaccine by choosing the prevalent serotype ofP. aeruginosa G,B and Klebsiella pneumonia K1as the antigens. Compared to thetraditional adjuvant Al(OH)3, Klebsiella pneumonia is not only able to keep the hostfree from infection as vital antigen in the vaccine but also has a good adjust functionto enhance the host immune response to P. aeruginosa. This leads high production ofantibody titer and offers better protection against pathogens.