| Mink hemorrhagic pneumonia is an acute and septic infections caused byPseudomonas aeruginosa (P. aeruginosa), and its main symptoms is hemorrhagicpneumonia and sepsis. The epidemiology in local region is important for theprevention and treatment against mink hemorrhagic pneumonia. P. aeruginosa has ahigh level of intrinsic resistance to a wide array of antibiotics and is cross-reactivitywith a variety of pathogens because of OPE. And it has no national certificationvaccine against mink hemorrhagic pneumonia in China. So the prevention, treatmentand diagnosis to this disease are difficult. In order to protect minks from minkhemorrhagic pneumonia in Shandong, the epidemiology was investigated, a rapiddetection by colorimetric loop-mediated isothermal amplifcation (LAMP) assay wasestablished and a trivalent inactivated vaccine against mink hemorrhagic pneumoniawas generated and evaluated in this study.90isolates of P. aeruginosa were isolated and identified from132hemorrhagicpneumonia minks, accounting of68.1%of total. The90isolates of P. aeruginosa wereclassified into four serotypes (sevovar G, B, C, I) using the slide agglutination methodaccording to the Homma schema using sera obtained from DENKA SEIKEN CO.,LTD.; serotype G was the most frequent (50/90,55.6%). Serotype C was found in18isolates, serovar I was found in17isolates and serovar B was found in5isolates. Witha diversity index of0.96,43genotypes (1~43) were identified among90isolatesexamined by ERIC-PCR. The most prevalent ERIC-PCR types were type18, found in17.8%of the outbreaks, and type39found in16.7%of the farm outbreaks. Theremaining genotypes contained1-3isolates each. Most of isolates were sensitive toenrofloxacin.Then, in order to established a detection of P. aeruginosa by colorimetric LAMPassay,3pairs of primers were designed according to the ETA gene of P. aeruginosa,including a pair of outside primers F3/B3, a pair of internal primers FIP/BIP and apair of loop-primer LF/LB. The hydroxyl naphthol blue was added in the reactionsystem. And the best temperature of amplification was65℃, the best optimum timewas30min. The sensitivity of this assay was10-fold than PCR, and had nocross-reaction with Escherichia coli, Pasteurella multocida, Staphylococcus aureus and Klebsiella. All90isolates could be detected by this assay. It indicated that thecolorimetric LAMP assay could be used to rapid-diagnosis of mink hemorrhagicpneumonia.According to the epidemiology in Shandong, three candidate isolates wereselected to develop trivalent inactivated vaccine, including of serovar G strain SD312,serovar C strain SD302and serovar I strain SD403. The virulence and bacteriacontent of these strains all were stable in30continuous passages. Three batches oftrivalent inactivated vaccine had been prepared in the best culture conditions. Thetrivalent inactivated vaccines were detected by sterile test, safety inspection and theinspection of the formaldehyde and mercury content and were safety.Subsequently the trivalent inactivated vaccine was evaluated by immunizingmice and mink. The minimum immunizing dose for mink was0.25mL, and theimmunizing dose was1mL. The antibody titer peaked on21~28days post-immunizedand began to decline on42days post-immunized. The weight of mice immunized bytrivalent inactivated vaccine had no significant decline (P>0.05), compared with theno-immunized mice. Immune protection time of the vaccine was180d. Field trialsshowed that the trivalent inactivated vaccine had a positive effect on the prevention ofmink hemorrhagic pneumonia in Shandong.In conclusion, the present study provides the epidemiology of mink hemorrhagicpneumonia in Shandong, a rapid detection assay of colorimetric LAMP for P.aeruginosa and a candidate vaccine against mink hemorrhagic pneumonia. It may laysolid foundation for the prevention, rapid-diagnosis and treatment of minkhemorrhagic pneumonia. |
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