| Mycoplasma ovipneumoniae?Mo?is the main pathogen which causing respiratory infection and atypical pneumonia in sheep and goats.It has caused a great economic loss to the worldwide sheep industry.Vaccination is the most effective way to prevent the sheep from infecting Mo.However,Mo is difficult to cultivate,so it is the main reason for restricting the development of the vaccine.Therefore,the problem of how to improve the growth titer of Mo is urgently to solve.In China,Mycoplasmal pneumonia of sheep and goats?MPSG?is mainly caused by Mycoplasma ovipneumoniae?Mo?and Mycoplasma mycoides subsp.Capri?Mmc?.However,Mmc is easy to cultivat and it has a higher growth titer compared with Mo.In this study,the HiSeq sequencing platform of Illumina company was used to analyze the Mo and Mmc at different growth stages by transcriptional sequencing?RNA-seq?and bioinformatics analysis.RNA samples were collected at the logarithmic,stationary,decline from Mmc PG3 strain and Mo NM-151 strain,respectively.A point-to-point comparison between the corresponding phases of PG3 and NM151 strain was performed.Finally,the basic reasion of Mmc is easy to cultivate than Mo at the gene level can be find out.Hypothesis was proposed and validated based on the key requirements for Mo to fill the metabolic maps,which is significant to Mo culture and application.1.The analysis and comparison of the transcriptional sequence of the Mo NM-151isolate and the Mmc PG3 standard strain by the KEGG PATHWAY and the RPKM value of some genes in the pathway.It was found that the glucose metabolism and the nucleotide metabolism pathway may be the key pathway affecting the growth and metabolism of Mo.Besides,the Glucose?Glc,G?or Serin?Ser,S?was found,their addition may promote the growth of Mo.2.Based on above,add Glucose and Serin respectively to the Modified Hayflick's culture,which were optimized and further proved via culture test:the eighty-third generations of the NM-150 strain was incubated in four media?1:20?for 240 h,bacterial suspensions were collected every 12 h to make the growth curve of Mo using Colony-Forming Unit?CFU?,pH values and OD630nm30nm values.?1?By adding Glucose and Serin components directly to the control medium?MH?,three kinds of medium,G-MH,S-MH and GS-MH were formed to verify the culture.The results showed that the growth of the three medium had no significant effect on the growth of Mo.?2?It infers that the above results may be due to the low pH value,which leads to inactivation of enzymes during anabolism.Therefore,seven kinds of buffer systems were screened.The results show that the 20 mmol/L HEPES buffer system has the best effect.It can effectively decrease the pH value and increase the growth titer from 108 CFU/mL to 109CFU/mL,and prolong the stable period of Mo.?3?20 mmol/L HEPES was respectively added to the above medium of?1?to form three kinds of medium,which are GH-MH,SH-MH and GSH-MH.The results showed that total medium could increase the number of Mo bacteria,GSH-MH had no significant effect.GH-MH could greatly increase the growth titer of Mo,and the effect was best in increasing the number of bacteria,and SH-MH could also increase Mo by an order of magnitude.But the most notable feature is that it can significantly prolong the growth cycle of Mo.?4?The comparison test of GH-MH and SH-MH before and after the initial pH value was carried out.The results showed that the increase of the initial pH value could effectively slow down the decreasing speed of the pH value of the culture medium,stabilize the pH value at a higher level,and improve the living bacteria content and total bacteria amount in the medium,which further indicated that the pH value was One of the key factor affecting the growth of Mo.To sum up,transcriptomics bring a new direction for the study of mycoplasma,which lays an essential foundation for further study of Mycoplasma ovipneumoniae,and provides a certain theoretical basis for the development and production of Mycoplasma ovipneumoniae inactivated vaccines. |
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