Isolation And Identification Of Pseudomonas Aeruginosa From Mink In Shandong Province And Immune Efficacy Evaluation Of Tervalent Inactivated Vaccine In Mice

Mink hemorrhagic pneumonia is an acute mink respiratory infectious disease caused by Pseudomonas aeruginosa and its main symptoms are lung congestion and sepsis. It harmed the mink industry because it has some characteristics like acute occurrence, high mortality, local epidemic et al. With the increasing of drug-resistant strain and the pathogen strains are more widespread, drug treatment are more poor after the onset of infection in mink.The main prevention measures of the disease are employing inactivated strains whose serotypes as same as pathogenic strains.Therefore, bacterial isolation and identification in mink hemorrhagic pneumonia epidemic area is the basis and prerequisite for the preparation of multivalent inactivated vaccines as same as serotype survey. The contents of this study include bacterial isolation and identification in mink hemorrhagic pneumonia epidemic area, pathogen serotype distributional investigation and immune efficacy of trivalent inactivated bacteria in mice.Bacterial isolation and identif ication. During 2013 to 2014, our laboratory isolated 120 strains from 77 mink lung samples which come from 18 mink farms like Zhucheng, Weifang, Linyi, Wendeng, Rizhao, Juxian, Jiaonan, et al. Using biochemical tests and 16 S r DNA PCR identification ultimately determine the 53 strains of isolated bacteria were Pseudomonas aeruginosa. The isolation rate of Pseudomonas aeruginosa was 44.2%(53/120). The 53 isolates of P. aeruginosa were classified into 11 serotypes and an undetermined strain using the slide agglutination method. Serotype G was the most frequent(27/53). Serotype B was found in 5 isolates, serotype D was found in 4 isolates. Thus the most frequent serotypes were serotype G, serotype B and serotype D.Selection of immunogen. We preparated monovalent vaccines by mixing adjuvant with 15 inactivated strains which included 11 serptypes and immuning rabbits which agglutination test are negative. Three immunizations c ould make us obtain 11 kinds positive serums of Pseudomonas aeruginosa. Each antiserum was challenged against 11 strains using slide agglutination test. The results showed that the serum G-type, B-type, D-type and N-type had higher cross-reactivity than others. According to serotype identification results and serological cross-protection results, the mink hemorrhagic pneumonia inactivated vaccine serotypes are serum G-type, B-type and D-type. Through the screening of virulent strains, half lethal dose test and growth curve test, the alternative vaccine strains were SD004, SD050 and SD052.Inactivated vaccine immunogenicity detection. As the stability of SD004, SD050 and SD052 were determined, the candidate strains were continuously cultured in the same order of magnitude during twenty generation as same as the pathogenicity.The virulence and bacteria content of these strains all were stable in continuous passages. The three candidate strains including medium, culture temperature, culture time, culture medium p H, fermentation conditions were optimized and found the condition of 37℃ under p H7.0~p H7.5 broth medium culture 22-24 h for vaccine production is appropriate; Subsequently the trivalent inactivated strains was evaluated by immunizing mice. Respectively using four different immune doses with the same cell concentration of Kunming mice to immune, determined the minimum immune dose was 0.15 m L(1.125×1011CFU) that can protect against infections in mice with 10 LD5 0 of bacteria liquid poison attack after 21 days; The observation of the lung pathological changes in mice showed that the enhancement of the immune vaccine dose could protect the mice effectively and prevent the infection of Pseudomonas aeruginosa. The antibody titer peaked on 28 days post-immunized and began to decline on 42 days post-immunized. We could also detect antibodies at 49 day. The results of the two immunization of mice were higher than once. The protective effect was better, which could effectively reduce the possibility of infection of Pseudomonas aeruginosa in mice. After immunization 21 days we immuned candidate strains with live strains, the mice didn’t die and were propected completely by inactivated strains. The observation of lung, liver and kidney pathological changes in mice show ed that there were not pathological changes in mouse viscera, it proved that trivalent inactivated strains could protect the mice effectively.This study showed that we isolated predominant strains by serological investigation and biological characteristics detection with good immunogenicity which induced good immune response in mice, which could be candidate vaccine strains to prevent mink hemorrhagic pneumonia epidemic.