Identification Of Serotypes And Distribution Of Virulence Genes In Pseudomonas Aeruginosa Isolated From Mink And Its Pathogenesis In Mink

Pseudomonas aeruginosa(P.aeruginosa)thrives in moist and wet conditions and is able to utilize a wide range of organic compounds,which is a prevalent opportunistic human pathogen.Hemorrhagic pneumonia caused by P.aeruginosa remains one of the most costly infectious diseases among farmed mink and commonly leads to large economic losses during mink production.In this study,the serotypes and vir?Lence genes of P.aeruginosa were tested and their pathogenicity was studied.These studies provide important molec?Lar epidemiological data for the prevention and monitoring of P.aeruginosa.There are important implications in clinical practice and future research.From June 2016 to September 2016,a total of 112 were collected from Zhucheng,Liaocheng,Linyi,and other places in Shandong,and the bacteria were isolated and identified by routine bacterial isolation and culture techniques.There are 20 strains of suspicious colonies that can be selected based on the pigmentation of colonies,the presence of aromatic odors,and the ability to grow at 42 degrees and not grow at 4 degrees.And further confirmed by the amplification of 16S rRNA gene,the homologous relationship between the 16S rRNA gene sequence of the gene sequence alignment and the published organism in GenBank was studied.BLAST analysis in NCBI,further confirming that these are indeed P.aeruginosa.A total of 14 serotypes(A-N)were detected in 20 strains of P.aeruginosa using the slide agglutination test method.The Pseudomonas aeruginosa diagnostic serum kit was used.Eighteen of the 20 P.aeruginosa isolates belonged to serotype G using slide agglutination,one serotype B and oneserotype C.P.aeruginosa-CS-11 and P.aeruginosa-CS-20 were isolated from the same mink,but P.aeruginosa-CS-11 belonged to serotype B and P.aeruginosa-CS-20 belonged to serotype G.Interestingly,P.aeruginosa-CS-11 and P.aeruginosa-CS-20 were isolated from the same mink,but P.aeruginosa-CS-11 belonged to serotype B and P.aeruginosa-CS-20 belonged to serotype G.90%of the P.aeruginosa isolates belonged to serotype G using slide agglutination,5%serotype B and 5%serotype C.The 12 virulence-associated genes of the P.aeruginosa isolates were tested using PCR and sequencing.The sequence analysis demonstrated that the prevalence of the algD gene for the isolates was 95%(19/20),the plcH gene 85%(17/20),the exoY gene 80%(16/20),the aprA gene 75%(15/20),the lasB gene 70%(14/20),the exoS gene 65%(13/20),the exoT gene 60%(12/20),the exoU gene 0%(0/20),and the toxA gene 60%(12/20),the lasA gene70%,the phzS gene 60%,the phzM gene 55%.Our results indicate that all these virulence genes(exoT,plcH,algD,aprA,exoY,exoS,toxA,lasB,lasA,phzS,phz M)are major in mink infection.Phylogenetic analysis and homology analysis showed that Pseudomonas aeruginosa separated from mink is closely related to human origin,potentially at risk of human infection.To compare the difference in pathogenicity between different serotypes,P.aeruginosa-CS-2and P.aeruginosa-CS-10 strains were selected for animal experiments.The LD50 of the P.aeruginosa-CS-2 was 6.8×10~4colony forming units(cfu).The LD50 of the P.aeruginosa-CS-10was4.6×10~5colonyformingunits(cfu).Thevirulenceof P.aeruginosa-CS-2 in mink was higher than that of P.aeruginosa-CS-10.Lung tissue taken from a mink died characterized by thickening of the alveolar septa.Liver tissue taken from a mink died characterized by congesting and steatosis.Spleen tissue taken from a mink died characterized by pyknosis and bleeding.Heart tissue taken from a mink died characterized by hemorrhage.The control mink showed no clinical signs.Mink(P.aeruginosa-CS-2)first showed clinical signs.Meanwhile,P.aeruginosa-CS-10 did not cause any clinical symptoms.It is might be partly due to the virulence gene content difference between P.aeruginosa-CS-2and P.aeruginosa-CS-10,especially Exotoxin A(toxA).ToxA is an important virulence factor of P.aeruginosa in clinical infections and is the most virulent factor among other factors produced by P.aeruginosa.