| Mycoplasma pneumoniae(MP)is the main pathogen of the common community acquired pneumonia(CAP)in children,which can cause atypical pneumonia and a series of complications.The aim of this study was to isolate and cultivation Mycoplasma pneumoniae strains from clinical samples,and to screen the MP isolates with strong protection and safety as vaccine candidates,and lay the foundation for the development of Mycoplasma pneumoniae vaccine.In this study,the medium for isolation of MP was screened by comparing the effects of different medium and additions on the growth rate of MP and the effect of CCU.On the basis of the selected SP-4 medium,different antibiotics were added and filter was used to remove the other microorganisms.The enrichment time was explored to establish the isolation and cultivation method of Mycoplasma pneumoniae.The results showed that with 20%horse serum as addition,the SP-4 culture as base,the 0.5%glucose could make MP grow best,fast and more yield,and then add the final concentration of 500 U/mL penicillin G,2.5 U/mL polymyxin B,2.5 ?g/mL amphotericin and 0.0025%thallium acetate,which can effectively inhibit the growth of the other microorganisms,but not obviously inhibit the growth of MP;After that,most of the bacteria were removed by 0.45 ?m and 0.22 ?m filter and the enrichment of swabs and sputum samples could be removed.The best separation time was 6-8 h and 18-24 h respectively.In this study,30 strains of Mycoplasma pneumoniae were isolated from 689 children's swabs and sputum samples.After RFLP typing,25 of them were I and 5 were II.In order to screen the MP isolates with good immunogenicity and low virulence,we sequenced the main antigen genes(p1?p30?p65)and virulence related genes(cards,mpn491)of all the isolates,and selected 11 representative strains to detect its pathogenicity in Balb/c with 108 CCU/mL(× 100 ?L)by intranasal immunization in 2 days,protective ability was 107 CCU/mL(× 100 ?L)intranasal immunity to Balb/c mice for 1 times,and 21 d after 108 CCU/mL(× 100 ?L)with the same standard strain.The pathogenicity and protective ability of MP isolates were analyzed from the wet lung index and the lung histopathological point of view.Pathogenicity test found that the pathogenicity of isolate(No.)14 was the weakest,the rate of body weight change of Balb/c was 5.73± 1.17%,the wet weight lung index was 0.93±0.10%,isolate(No.)21 and 27 were the strongest.The rate of body weight change of mice infected with Balb/c was-16.95 ±4.02%,-14.76± 11.31%,wet heavy lung The number was 1.44±0.06%;1.46±0.11%;the pathological score of lung tissue in mice was]2± 7;12±2,and there was no significant difference between two(P>0.05).The results showed that the protective effect of type ? isolates after intranasal immunization was better than that of isolates I,in which the isolate(No.)14 was immunized with FH strain and the wet weight lung index of mice was 0.71 ±0.07%,and the lung histopathology score of mice was 3 ±1,which was significantly better than that in the positive control group(P>0.05)it shows the best protection effect.This study established a method of isolation and cultivation of MP strains from clinical specimens,and successfully isolated 30 strains of MP strains,of which isolate(No.)14 was the weakest,the best protective force,and the isolate(No.)21 and 27 had the strongest pathogenicity.This study could lay the foundation for the selection of candidate strains of Mycoplasma pneumoniae vaccine and develop the vaccine.It has important application value. |
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