Screening, Induction And Biological Characteristics Of Corynebacterium Pseudotuberculosis Attenuated Strain

Corynebacterium pseudotuberculosis is a Gram-positive bacteria that can cause disease in small ruminants including sheep and goats,and cattle,horses,camels,pigs and other animals.It is an important pathogen of Caseous lymphadenitis?CLA?,a chronic infectious disease.CLA epidemic in most areas of the world's flock.Once infected with C.pseudotuberculosis in the flock,it is difficult to control and eradicate the pathogen,and causing great economic losses to the sheep industry.In addition,the infection of C.pseudotuberculosis can also cause lymphadenitis,pneumonia and endocarditis in humans,which has a potential threat to human health.Therefore,there is an urgent need to study prevention and control schemes that can effectively reduce the infection of the pathogen.At present,the main methods to treat C.pseudotuberculosis are drugs and surgery in China.Since the cystic membrane of the abscess is thick,and the pus in the cyst is thick,the drug treatment effect after the onset is not ideal.Surgical treatment needs to be performed on each sick goat,and the treatment efficiency is low,and the environment is easily polluted when discharging pus.Therefore,the most effective way to control the disease is vaccine immunization.Although many regions of China have reported the infection and epidemic of C.pseudotuberculosis,there is currently no commercial vaccine available in China.The live vaccine has the characteristics of rapid immunity generation,long immunization duration,good immunization effect and low cost..Based on the screening of 4 strains with weak pathogenicity from the clinical isolates of C.pseudotuberculosis infected with goats in the early stage,this study repeated pathogenicity tests and immunoprotection tests of selected strains to choose candidates for attenuated vaccines with low pathogenicity and good immunogenicity.Furthermore,the selected strains were subcultured and weakened by means of physical mutagenesis,chemical mutagenesis,and compound mutagenesis.The aim was to cultivate candidate vaccine strains with low pathogenicity and good immune effect,which laid the foundation for the development of C.pseudotuberculosis attenuated vaccine..The results are as follows:1.Screening of vaccine candidate strains of C.pseudotuberculosisFour clinical C.pseudotuberculosis isolates,namely Dazu strain?DZ6?,Fuling strain?FL2-5?,Yunyang strain 2?YY2?and Yunyang Strain 6?YY6?,with relatively weak pathogenicity,were used for repeated pathogenicity tests and immunogenicity tests on mice.The results showed that the average survival time/survival rate of Kunming mice inoculated with the bacterial solution?OD450=0.08?were 187.4 h/20%?YY2?,228.4 h/40%?FL2-5?and 318.8 h/80%?DZ6?,and YY6 has no data due to culture contamination.The immune protection rate against 4×LD₅₀ C.pseudotuberculosis Wanzhou?WZ?virulent strain is as follows:50.00%?YY2??66.67%?DZ6??33.33%?FL2-5?and 16.67%?YY2?.It is suggested that the DZ-6strain of C.pseudotuberculosis has lower pathogenicity and bwtter immunogenicity than other strains,and is determined as the candidate strain of attenuated vaccine for subsequent research on the pathogen.2.Attenuation and biological characteristics of candidate strains of C.pseudotuberculosis attenuated vaccineDZ6 were subcultured with physical mutagenesis?ultraviolet rays,higher temperature?,chemical mutagenesis?nitrite action?and compound mutagenesis?ultraviolet light+nitrate action?.The effects of different attenuate methods on the morphology,growth characteristics,pathogenicity and immunogenicity of DZ6colonies were observed.The results showed that the ultraviolet exposure time was 1min,and the lethal rate for C.pseudotuberculosis was 86.5%,the nitrite effect time was 3 min,and the lethal rate for C.pseudotuberculosis was 83.1%,while cultured at41?for 24 h,the lethal rate for C.pseudotuberculosis was 83.3%.Except for the faster growth of subcultured 20^th generations of DZ6 than the primary strain in 8 h to24 h,,there was no significant difference in colony morphology and staining characteristics betweet subcultured 20^th generations of DZ with mutagenesis and the primary strain.The lethal rates of primary generation and 20^th generations of DZ subcultured under regular condition,ultraviolet mutagenesis,ultraviolet mutagenesis?larger colonies?,nitrous acid mutagenesis,ultraviolet+nitrous acid mutagenesis and41?to mice?1×10⁶ CFU/0.2 ml?were 60.0%?60.0%?53.5%?33.3%?60.0%?40.0%and 20.0%respectively.Compared with the primary strains,the spleen bacterial load and the number of abscesses of the 20^thh generation subcultured at41?were significantly decreased,and the liver bacterial load was significantly reduced.Compared with the primary DZ6 strains,the spleen bacterial load and the number of abscesses of the 20th generation strains subcultured at 41?were significantly decreased,and the liver bacterial load was significantly reduced.There was no significant difference in other mutagenized subculture strains,and there was no significant difference in spleen index of each mutagenized subculture strains;The immune protection of DZ6 and its 41?mutagenized 20^thh generation strain against 10×LD₅₀ Wanzhou virulent strain?WZ?was 40.00%?primary strain DZ6?and 60%(Mutagenized DZ6 strain subcultured 20^th generation at 41?,DZ6WD-20).The results indicated that the subcultivation by mutagenesis at 41?can reduce the pathogenicity of DZ6 without any significant effect on its immunogenicity.3.Preliminary study on the mechanism of C.pseudotuberculosis virulence attenuationIn order to explore the mechanism of high temperature mutagenesis to reduce the virulence of the strain,the expression of phospholipase?PLD?,sigma E factor?sigE??small fimbriae?SpaC?,neuraminidase?NanH?,threonine protein kinase G?PknG?and superoxide dismutase?SodC?were det alected in 20^th generation of DZ6 subcultured at 41??DZ6WD-20?using real-time fluorescence quantitative PCR method.The sequence of PLD,the main virulence gene of C.pseudotuberculosis,were comopared between DZ6 and DZ6WD-20.The results showed that the expression of the virulence genes NanH and SodC ofDZ6WD-20 was significantly lower than that of the primary Dazu strain?DZ6??p<0.05?.The expression of the virulence genes PLD,sigE,SpaC and PknG were also significantly reduced?p<0.01?.The sequencing results showed there was no change in PLD gene sequence between DZ6WD-20 and DZ6.It is suggested that the decreased pathogenicity of DZ6 induced by culture at41?may be related to the down-regulation of PLD,sigE,SpaC,NanH,PknG and SodC virulence factors of C.pseudotuberculosis.In summary,this study screened and obtained a vaccine candidate strain of C.pseudotuberculosis?DZ6?;It was found that passage at 41?could reduce the pathogenicity of DZ6 strain without affecting its immunogenicity;The mechanism of reducing virulence of may be related to the down-regulation of PLD,sigE,SpaC,NanH,PknG and SodC expression in DZ6subcultured at 41?.The results of this study laid the foundation for the further weakening of DZ6 strain and the development of attenuated vaccine against C.pseudotuberculosis.