Identification And Pathogenicity Test Pseudomonas Aeruginosa From Mink

Along with the economy development, fur animal breeding industry in Shandongis growing rapidly. Shandong Province has become the No.1 fur animal-feeding province in China, of which the number of fur animals raised is about 50% of that of country. However,with the increasing intensivism in fur animal-breeding industry, the incidence of related fur animal disease has been increasing. The situation of prevention and controlling in epidemic disease is serious. Epidemic disease is the important factors to restrict the development of fur animal-breeding industry, which caused great economic losses to breeders and enterprises. Mink hemorrhagic pneumonia is one of serious mink diseases which block the healthy development of the industry and become increasin gly serious. Pseudomonas aeruginosa is one of the most major pathogen which causes mink hemorrhagic pneumonia. In order to effectively prevent and control mink hemorrh agic pneumonia, it’s necessary to investigate epidemic state of Pseudomonas aeruginosa in mink, especially clarify the pathogenicity of Pseudomonas aeruginosa to mink.Isolation and identification of Pseudomonas aeruginosa were performed from 43 lung tissue samples by conventional techniques.Selected representative isolate to establish animal models of pathogenicity of P.aeruginosa and clarify the pathogenicity of Pseudomonas aeruginosa.1. Isolation and identification of Pseudomonas aeruginosaBacteria isolation were performed from 43 lung tissue samples which were collected from mink with hemorrhagic pneumonia from Zhucheng, Wendeng,Linyi,et al in 2013. The isolates were identified by morphological observation, physicochemical test, drug susceptibility test and 16 SrDNA sequencing et al. As a result, the 5 isolates were all Pseudomonas aeruginosa,named F1, F5, F6, F8 and F10, and isolating rate was only 11.6%.Drug-sensitivity tests indicated that the 5 isolates were resistant to Penicillin, Erythromycin and kanamycin. 5 strains was 80.9 %~100% homological to the14 reference strains and the nucleotide homology of 5strains was 98.9 %~99.7 % by 16 SrDNA sequence homology analysis. Phylogenetic analysis showed that F5、F6、F8、F10 and F1 were clustered in the same branch.2. Pathogenicity test of Pseudomonas aeruginosa from minkF1 was selected as representative strain for challenge test of mice, and animal models of pathogenicity of P.aeruginosa to mice was established. 30 healthy mice were divided into six groups, 5 for each group. The six groups of mice were kept in separated rooms of the isolation facility. The mice in group 1-5 were inoculated intraperitoneally with the bacteria solution of F1 respectively of which CFU was 6.4×107、6.4×106、6.4×105、6.4×104、6.4 ×103, respectively.Group 6 was negative control. Clinical signs of mice were monitored within 7 days after inoculation. Autopsy for dead mice during the test period,at the same time,the following tissue samples were collected from dead mice for histopathologic examinations and bacterial load detection, including heart, liver, spleen, lung, kidney.As a result, the mice of group1 and 2 were all dead within 12-18 h. The characteristic changes of organs at necropsy were varying degrees of bleeding, especially lung lesion was more serious.Pathohistological examination under microscope showed that myocardium hyperemia,hepatocyte necrosis, a large number of inflammatory cell existed in spleen, pulmonary congestion, alveolar walls thickened, erythrocyte existed in renal interstitium. P. aeruginosa were isolated from all of these organs. The bacterial load in the lung was higher than in other organs,while in the spleen was lower. The LD50 for mice was 1.6×106CFU/mL. The result of pathogenicity test of P.aeruginosa in mice indicated that strain F1 had stronger pathogenicity to mice.F1 was also selected as representative strain for challenge test of mink, and animal models of pathogenicity of P.aeruginosa to mink was established. 20 healthy mink were divided into 5 groups, 4 for each group. The 5 groups of mink were kept in separated rooms of the isolation facility. The mink in group 1-4 were inoculated intranasally with the bacteria solution of F1 respectively of which CFU was 1.6×108、 1.6×106、 1.6×104、1.6×102respectively. Group 5 was negative control. Clinical signs of mink were monitored within 14 days after inoculation. Autopsy for dead mink during the test period and the mink which were euthanized at the end of the experiment, at the same time the following tissue samples were collected from dead mink for histopathologic examinations and isolation of P.aeruginosa, including heart, liver, spleen, lung, kidney. As a result, the mink of group1 were all dead within 20-44 h. The characteristic changes of organs at necropsy were pulmonaryhemorrhage and splenomegalia. Pathohistological examination under microscope showed that myocardium hyperemia, hepatocyte degeneration, splenorrhagia and spleen necrosis,pulmonary hemorrhage. P. aeruginosa were isolated from heart, liver, spleen, lung, kidney of dead mink. The LD50 for mink was 3.2×107CFU/m L. The mink in other artificial infection groups only showed transient depression and anepithymia on 1-3 day post infection, and clinical symptoms gradually disappeared on 4 day post infection until at the end of experiment. Pathohistological observation on the mink in group2-4 and negative control showed no pathologic changes. Furthermore, P. aeruginosa were not isolated neither from the mink in group2-4 nor negative control.The result of pathogenicity test of P.aeruginosa in mink indicated that P. aeruginosa can infect mink, but pathogenicity is lower, there were still other pathogens can cause mink hemorrhagic pneumonia. Our study clarify the pathogenicity of Pseudomonas aeruginosa to mink, which will provide the experimental data and useful theory support for the prevention and control of mink hemorrhagic pneumonia.