Immunoprotection Of The Inactivated Vaccine Against Streptococcus Agalactiae Infection For Tilapia And Characteristics Of XtgS Gene Deletion

S.agalactiae is a class of Gram-positive cocci,classified as B group according to the serum classification.The strain is P hemolysis and mostly long chain.As a zoonotic pathogen,it can not only infect newborns and non-pregnant adults.In recent years,large-scale S.agalactiae infection in tilapia farms caused large economic losses on aquaculture industry.China is a big country in the aquaculture production.The demand of people for aquatic products is growing.The pathogenicity of S.agalactiae increases the difficulty to control,due to its multi-factor,multi-channel and multi-mechanism and so on.The S.agalactiae infection can be prevented and controlled by the effective vaccine with the maximum immunogenicity and minimal side effects.Studying in the unique virulence island of the pescine S.agalactia GD201008-001 contributes to clarify its pathogenesis.1 Immunoprotection of the Inactivated Vaccine against Streptococcus agalactiae infection for Tilapia.After the S.agalactiae GD201008-001 isolated from tilapia was continually passaged by 50 generations,the biochemical characteristics of every passage were compared by API 20 Strep and automatic biochemistry analyzer.30g ± 5g tilapias were immunized by IM injection and IP injection with 5 x 108 CFU/fish of inactivated vaccine.50 LD50 challenged the tilapias 14 days after three immunizations and the death of tilapia was recorded.The immunological protective efficacy on tilapia was analyzed by detecting some enzyme indexes of livers with kits and the expression of IL-1? in head kidney with RT-PCR at different time points during the immune phase.The biochemical characteristics were stable among different generations.The RPS values for these groups were 83.97%and 93.33%post infection.The inactivated vaccine is effective in protecting tilapia against high concentration of S.agalactiae,so that it can be used as a vaccine candidate against S.agalactiae.There was no significant difference among the most enzyme indexes with different injections before and after immunization(P>0.05).The IL-1? expressed significantly higher at 42 days than 14 days after the first immunization(P<0.05)and the expression of the IL-1? was significantly higher by IM injection than IP injection(P<0.05).The SOD,LZM,T-AOC,CAT immunized by IM injection showed slightly lower than by IP injection,which was contrary to the MDA.It is indicated that intraperitoneal injection of inactivated vaccine is more effective.2 Construction and biological characteristics of XtgS gene delation in Streptococcus agalactiae GD201008-001.From the previous study,the specific lOkd genes island in the pescine S.agalactiae GD201008-001 contains a DNA binding protein XtgS,whose function was never researched before.So in this study,the XtgS gene was knockout without trace.The deletion plasmid pSET4s-XtgS was constructed by the method of homologous recombination and the using of the streptococcus-Escherichia coli shuttle vector pSET4s.The plasmid was introduced into GD201008-001 by electroporation.The ?tgS generated via double-crossover was selected in the presence of the appropriate antibiotics and temperature.The mutant strain was verified by the mRNA level.The results showed that the ?XtgS showed no significant difference in the bacterial morphology,the minimum inhibitory concentration of some common antibiotics and adherence to HEp-2 and bEnd.3 cells compared with the wild-type strain,but the growth rate was slightly slower than the wild strain GD201008-001.Both in vivo and in vitro ICR mice model,the proliferation of the AXtgS was faster than the wild strain in a short time,but the content and distribution was close to each other before death.Among the brain and blood,the bacterial content was the higher in the blood than the brain.The expression of inflammatory cytokines TNF-? in serum was little higher than that in wild type groups.The results showed that the ?XtgS was successfully constructed and the virulence of bacteria was enhanced to some extent,which may be a negative transcriptional regulator regulating the expression of virulence genes of S.agalactia GD201008-001,although its mechanism required to be further confirmed.3 Characteristics of the pescine Streptococcus agalactiae GD201008-001 and the human strain A909.Comparing the genome analysis,the genome sequences and genetic elements of the pescine strain GD201008-001 and the human strain A909 were highly similar.The two strains could be analyzed from several angles.In this study,we compared the two strains preserved in the laboratory and conducted a series of experiments to compare the differences in biological characteristics,such as bacterial morphology,biochemical status and growth curve.The survivals of the two strains in the blood of the mice and tilapias were carried out to compare the virulence,following the LD50 of the zebrafish infected model.The mice were intramuscular injected by the two strains with the blank control.The samples stained with hematoxylin and eosin(HE)were taken at 0.5 h,1 h,3 h and 24 h post infection.There was no significantly difference in the bacterial morphology and biochemical characteristics,while the A909 growed faster and its LD50 was much larger than GD201008-001 with the significant difference(P<0.05).The survival rate of A909 was significantly higher than GD201008-001(P<0.05)except 1.5h in the blood of the tilapia.The results of HE staining showed that the exudation of inflammatory cells,blood cells,bacterias and the self-autolysis of the cells gradually increased with the passage of time.The phenomenons were more serious of GD201008-001 than A909.Although Similar in gene sequence,the pescine strain and the human strain are significantly different in virulence.The specific molecular mechanism of the differences should be further studied.