| Objective:Treponema pallidum is a highly invasive"stealing"pathogen that can rapidly spread from the infected site to multiple other distant tissues and organs through blood and lymph nodes,causing chronic and persistent systemic multiple-organ damage.The purpose of this study was to investigate the replication and proliferation of Tp in various organs of New Zealand rabbits and evaluate the immune protective effect of the infection-dependent antigen Tp0971Methods:1.Infecting the shaving New Zealand rabbits with purified Tp suspension by intradermal injection,closely observing the changes of skin redness and induration diameter at the site of inoculation,and recording the diameter of redness and induration,ulcer occurrence time and ulcer rate;2.Euthanizing three rabbits randomly at 3,7,14,21,35,65,100 and 130 days after infection,collecting blood,skin,testis,heart,liver,lung,spleen and kidney tissue.After extracting total DNA,the level of Tp DNA in each DNA specimen was detected by qPCR;3.Immunizing the rabbits with Tp0971 recombinant protein combined with CpG ODNs adjuvant,and set up CpG adjuvant control group and PBS control group.After immunization the Tp0971 specific IgG antibody level in serum were detected by ELISA;4.After last immunization,1mL1×10~7 T.pallidum purified Tp suspension was inoculated by intradermal injection.The changes of skin rash,induration and ulcer at the inoculation site were observed continuously for 30 days;5.Three New Zealand rabbits were randomly euthanized in each group on days 3,7,14,21,35,65,100 and 130 after Tp infection,and the level of Tp DNA in blood,skin,testis,heart,liver,spleen,lung and kidney were detected by qPCR;HE dyeing was performed after formalin fixation of the skins and testicular tissues,the infiltration of inflammatory cells was analyzed.Results:1.On the 4th day after the intradermal injection of Tp,the skin at the site of inoculation began to appear redness,hard chancre and ulceration appeared on the 6th day.On the 14th day,the rate of lesion and ulceration reached a peak(76.4%),on the 24th day,the skin ulcer at the inoculation site was basically healed;2.Within 14 days after infection,Tp was mainly concentrated at the skin inoculation site;On day 21,Tp was mainly concentrated in blood and hyperemic organs(heart,lung,liver and kidney).From 35th day after infection,the Tp load in blood and tissues were no significant difference;3.Tp burdens at the inoculation site of New Zealand rabbits remained at a high level(about 10~4 flaA copies per?g of rabbit DNA)for 21 days after inoculation;On the 35th day,it decreased to a lower level and then presented a slow increasing trend.Tp DNA was detected in other tissues and organs on the 3rd day after infection.On the 21st day after infection,the Tp load reached a relatively high level.At day 100 and day 130,the Tp DNA level in the organs of each tissue presented a state of slow increase or equilibrium;4.The recombinant protein Tp0971 was successfully induced and expressed,and the specific target band was identified by Western blot;5.The New Zealand rabbits produced a high level of Tp0971 specific IgG antibody,which were immunized with Tp0971/CpG recombinant protein,and the antibody level remained significantly higher than the CpG and PBS control group during the immunization period(P<0.01);6.Tp0971/CpG immunization can delay the skin ulcers occur and promote the healing of in New Zealand rabbits after syphilis infection,but which could not inhibiting the Tp spreading to other distal tissues and organs;7.Pathological analysis showed that the collagen fiber cells in the skin of the Tp0971/CpG immunized group were enlarged and proliferated and the dermis had a large number of chronic inflammatory cells infiltration;And there was no obvious infiltration of inflammatory cells in testicular tissueConclusions:1.New Zealand rabbits can present clinical symptoms similar to syphilis patients after being infected with syphilis,and the Tp load in all organs and tissues in the whole infection process presents a cycle of repeated and dynamic changes of decreasing and increasing;2.The Tp0971/CpG immunized rabbits could not completely eliminate Tp from the host body,nor could it inhibit the spread of Tp to other distal tissues. |
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