Studies On The Cytotoxic Effects Of Four Strains Of Haemo Philus Parasuis Isolated From Liaoning Province On PK-15

Haemophilus parasuis(HPS)is a gram-negative bacterium that colonizes the upper respiratory tract of healthy pigs.It can cause multiple fibrinous serositis and arthritis when the pig’s immunity is reduced or under stress.Meningitis and other typical pathological changes,this disease has now become one of the main diseases endangering the global pig industry.At present,there are more and more reports of HPS attenuated strains causing disease in pigs,which may be related to changes in virulence.In order to analyze the differences and changes in the virulence of virulent and attenuated HPS strains,in this study,the cytotoxicity of HPS attenuated strains 7 and 9 and virulent strains 5 and 13 isolated from Liaoning Province were analyzed to provide a scientific basis for revealing the pathogenicity of different serotypes of HPS,it also provides reference for prevention and control of Haemophilus Parasuis and healthy development of pig industry in China.Experiment 1.Study on the toxic effects of HPS strains with different virulence on PK-15 cells.Four strains of HPS were used to challenge PK-15 cells,and the viability of the cells was detected by cell pathological observation,MTT and LDH relative release tests,and the effects on cell adhesion and invasion rates were also tested to preliminarily determine the virulence of these four HPS strains The strengths and weaknesses.The results show that:(1)From the perspective of cell morphology,12 hours after HPS challenge,the PK-15 cells in the type 5 and type 13 challenge groups have uneven morphology,unclear edges of some cells,reduced adherent cells,and the disappearance of the original cell structure.The damage of type 7 and type 9 cells is mild,but some lesions may also appear;Transmission electron microscopy observed that 4 strains of HPS can cause certain damage to the microstructure of PK-15 cells.Among them,the damage ability of type 5 and 13 HPS is stronger than that of type 7 and 9 HPS.(2)After MTT detection of four HPS serotypes challenged HPS,the survival rate of PK-15 cells decreased(P<0.05 or P<0.01).Among them,the cell survival rate of type 9 HPS challenged was significantly higher than that of types 5 and 13 HPS(P<0.05),there was no significant difference between the two virulent strains and the HPS type 7 challenge group(P>0.05).(3)Compared with the type 7 and type 9 HPS challenge groups,the relative release of LDH in the type 13 HPS challenge group increased significantly(P<0.01),and the relative release of LDH in the type 5 HPS challenge group increased significantly(P<0.05),indicating that the virulent strains damage the cell membrane to a greater degree.(4)The adhesion ability of type 5 HPS is very significantly stronger than that of type 7,9 and 13 HPS(P<0.01);the intrusion ability of type 7 HPS is very significantly stronger than that of type 5,9 and 13 HPS(P <0.01),it is speculated that this may be one of the reasons for the increased virulence of clinical type 7 HPS strains.From the overall analysis,the virulence of HPS types 5 and 13 is still stronger than that of types 7 and 9.Experiment 2.Effect and mechanism of four HPS strains with different virulence in inducing apoptosis of PK-15 cells.The study used Annexin V/PI double staining,Western blotting and fluorescent quantitative PCR to study the apoptosis of PK-15 cells after challenge 12.The results show that:(1)Flow cytometry detected cell apoptosis.It was found that the virulent strains of HPS type 5 and 13 could induce apoptosis of PK-15 cells(P<0.01).HPS type 7 and type 9 were more capable of inducing cell apoptosis weak(P>0.05).(2)The relative expression of anti-apoptotic BCL-2 protein in the type 5 and type 13 HPS challenge groups decreased(P<0.05),and the relative expression of pro-apoptotic BAX protein increased significantly(P<0.05),indicating two The virulent strain can promote cell apoptosis through the BCL-2/Bax mitochondrial apoptosis pathway.(3)The relative expression of BCL-2 m RNA in type 5 and type 13 HPS challenge groups was reduced(P<0.05),the relative expression of BAX m RNA was extremely significant(P<0.01),while type 7 and 9 were compared with blank There was no significant difference between the two virulent strains and the virulent dwarf group,indicating that the apoptotic effect of the two virulent strains of HPS was achieved by up-regulating the relative expression of BAX m RNA and down-regulating the relative expression of BCL-2 mRNA.