【作者】 李晓云；

【导师】 袁宗辉； 毕丁仁；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2008， 博士

【摘要】 附红细胞体(Haemomycoplasmas,HMs)是一类无细胞壁、寄生在多种脊椎动物红细胞表面的微生物。附红细胞体引起的疾病即附红细胞体病(Eperythrozoonosis)简称附红体病,猪的附红体病临床上较为多见,对不同年龄段的猪均有危害,现已遍及世界各地,给全球养猪业造成了巨大的经济损失。由于缺乏体外培养系统,HMs研究进展缓慢。早年根据其部分生物学特性被分类为立克次体(Rickettsiales)。近年来研究发现HMs在系统进化树上与霉形体(Mycoplasma)的关系最为接近(76%～83%),且基因组大小(730～770 kb)、碱基组成特点(低G+C%)亦与霉形体类似,因而我们推测附红细胞体的营养需要、代谢特点可能与霉形体有相似之处,为了更详细观察和研究各种动物HMs的菌体形态、生理生化以及生长繁殖等特性,破解HMs在体外无细胞培养基中不能人工培养的难题,为HMs的临床血清学诊断、药物筛选及疫苗研制等提供理论基础,我们将猪等各种动物附红细胞体接种到通过配方筛选的霉形体培养基内,传代时不添加任何阴性红细胞,而以更换半量培养基的方式进行了体外培养。结果表明,动物附红细胞体在体外可长期传代并持续存活;皱缩的附红细胞体经培养可以恢复长大;将猪、羊附红细胞体从红细胞表面洗脱后亦能按上述方法用霉形体培养基进行连续传代并保持存活;但未能获得附红细胞体大量增殖的结果。在此基础上进行了病原体低温保存方法研究、抗生素敏感性试验,主要研究内容如下:1.猪、兔、犬、牛、羊附红细胞体的体外连续培养临床发生附红细胞体病猪血样,经PCR扩增和Giemsa染色镜检证明感染猪附红细胞体(Mycoplasm suis,M.suis)。经PCR扩增所得DNA片段,与M.suis已知序列同源性达99%以上。兔、犬、牛、羊血样若干份,经Giemsa染色镜检为HM阳性,红细胞染虫率(PPE)在50%～95%之间。经预试验对培养条件优化后,将猪、兔、犬、牛、羊等动物的HM阳性红细胞接种到多种培养基培养内,不添加阴性红细胞,每次传代更换半量培养基,结果发现经配方筛选的霉形体培养基PPLO-S-BS(PPLO Broth添加牛血清等成份)培养猪、兔、犬HM效果最佳,连续维持虫体(附红细胞体简称,下同)存活分别达280d、300 d、270 d以上,红细胞染虫率、染虫强度保持不变,虫体饱满光亮、活力较强;牛HM接种培养基后虫体皱缩,但经PPLO-S-BS等6种培养基培养虫体长大,并能维持其存活达120～150 d以上;PPLO-S-BS等8种培养基可维持羊HM存活达90～120 d以上。将M.suis、羊HM从红细胞洗脱后同法用PPLO-S-BS分别培养,培养至90 d、100 d左右后每5～10 d按1:2(v/v)的比例传代,累计传代各50次、45次,连续培养380 d后终止。通过对M.suis培养物PCR鉴定,对M.suis、兔HM、犬HM培养物的感染阴性红细胞试验,对M.suis、犬HM培养物扫描电镜观察,对所有培养物的光镜观察、抗生素敏感性试验,证实经长期培养的虫体与接种物为相同病原,所有传代附红细胞体一直处于存活并缓慢增殖状态。2.猪、兔、犬、牛、羊附红细胞体的低温保存研究将猪、兔、犬、牛、羊感染HM全血以柠檬酸三钠抗凝等方法处理后,于4℃保存,结果发现柠檬酸三钠抗凝后直接保存效果最佳,至少3 weeks后红细胞基本保持完整,染虫率不变,虫体存活状态良好,保存后皱缩的虫体经培养可以恢复长大。用改良低浓度甘油冷冻法等3种方法长期低温冻存猪、兔、犬、牛、羊感染HM的红细胞,结果发现改良低浓度甘油冷冻法冻存上述染虫红细胞效果最佳,复苏后红细胞存活率最高,PPE不变,HM保持有较好的活性。3.猪、兔、犬附红细胞体药物敏感试验有效药物作用于染虫红细胞后,虫体首先从红细胞上游离至培养液中,具有明显的自主运动,后运动减弱,虫体消失不见。红细胞染虫率、染虫强度不同程度下降,形态恢复正常,运动特点消失。用添加有强力霉素等8种抗生素的PPLO-S-BS分别对猪、兔、犬HM进行体外培养,结果发现,强力霉素杀灭效果最佳,强力霉素、土霉素杀灭和抑制HM的效果明显高于其他抗生素,可明显减小HM的PPE和PHE(p＜0.05)。泰乐菌素对HM有抑制作用,可明显减小HM的PHE(p＜0.05)和染虫强度,但对染虫率影响不大。更多还原

【Abstract】 Haemomycoplasmas(HMs) are well-less bacteria that parasitize the surface of erythrocytes of various vertebrate animals,and they are the causative agents of Eperythrozoonosis,in clinical cases,pig Eperythrozoonosis can be often seen and various aged pigs may catch Eperythrozoonosis.Up to now,porcine Eperythrozoonosis(PE) has spread all over the world and leaded to significant economic lost in the pig industry.The lack of an in vitro culture system prevents the further studying of these organisms. At the early years,according to their partial biology characteristics,HMs were classified to Rickettsiales.Current studies shows that HMs build a cluster of phylogenetically closely related to Mycoplasma and they share 76%～83%gene sequences similarity,similar genome sizes(730～770 kb)and base composition(lower G+C%),so we deduced that HM may share the similar nutrition requirement and metabolic characteristics with Mycoplasma.In order to further understand the morphology,physiological,biochemical properties,and growth and propagation characteristics of various animal HMs,This study aimed to solve the tough problem of HMs in vitro cultivation in cell-less medium,and thus build the theoretic foundation for serodiagnosis,drag screening and vaccine development of these organisms, HMs derived from various animals including pig were cultured using screened Mycoplasma based medium,and they were subcultured continuously using half fresh cell-less medium. The culture results indicated that animal HMs could be long-term in vitro cultured and survival in vitro,the shrank animal HMs could recover and grow up,but we did not obtain the results of rapid propagation of HMs.we did Antibiotics sensitivity test,low temperature in vitro conservation test were also carried out.The main study contents were reported as follow.1.In vitro culture of M.suis,rabbit HM,canine HM,bovine HM,ovine HMBlood samples collected from pig showing clinical signs of PE were positive for M.suis by Giemsa-stained blood smears and by PCR,the similarities of these amplified DNA fragments to the known sequence of M.suis(accession numbers AJ504999) were more than 99%.Blood samples derived from rabbits,dogs,goats were also proved to be HMs positive by Giemsa-stained,the range of percentage of parasitized erythrocytes(PPE) of HMs were 50%～95%.The optimal conditions of incubation for these positive blood samples were evaluated by preliminary test.Experiments were undertaken to develop in vitro cultivations of M.suis, rabbit HM,canine HM,bovine HM,ovine HM in various culture media with refreshing half volume of cell-less medium,results showed the optimal medium for M.suis,rabbit HM, canine HM was PPLO-S-BS(PPLO Broth supplemented with bovine serum,etc.) in which M.suis,rabbit HM,canine HM grew well,and this medium can be used to continuously culture M.suis,rabbit HM,canine HM for more than 280d,300d,270d respectively,after the culture,the PPE and the infected strength hold at the same level and HMs were very healthy and active.Bovine HM shrank severely in various media including PPLO-S-PS at first several days,then recoverd and grew up gradually.They could be maintained in these medium for more than 120～150d.Media PPLO-S-BS,PPLO-S-PS,PPLO-S-ES,FM4-BS, FM4-PS,M199-BS,RPMI1640-BS,DMEM-BS were similarly capable of supporting the continuous maintenance of ovine HM for more than 90～120 d.M suis and ovine HM were separated from erythrocytes and incubated with PPLO-S-BS, respectively.Approximately 90 d,100 d after culture,the subcultures were performed at split ratios of 1:2(v/v) at 5～10 d intervals(depending on the growth),respectively.These cultures were maintained for 380 d,with accumulative passages of 50,45,respectively,then the subculture were terminated.By PCR identification of M.suis culture,M.suis,light and electronic microscopic examination of M.suis and canine HM cultures,by infection of negative RBCs test of M. suis,rabbit HM,canine HM,and by the light microscopic observation and antibiotics sensitivity test of all cultures,we demonstrated that all tested animal HMs maintained survival and propagated slowly.2.Low temperature preservation at of M.suis,rabbit HM,canine HM,bovine HM, ovine HMSodium citrate anticoagulated whole blood samples of M.suis,rabbit HM,canine HM, bovine HM,ovine HM could be stored at 4℃for longer time than other methods,these samples can be stored at 4℃for at least 3 weeks,erythrocytes were integrity,and no changes of PPE were observed,parasites may shrank gradually,but they could grow up after incubating in PPLO-S-BS.Whole blood samples of M.suis,rabbit HM,canine HM,bovine HM,ovine HM can be long-term freezed by modified cryoapplication with low concentration glycerine method,it was the optimal method for cryopreservation of these animal HM,PPE and the survival rate of erythrocytes of all the samples did not changed after revival,all the organisms grew well, the infectivity to host erythrocytes,survival rate of erythrocytes were better than organisms cryopreserved by other methods.3.Drug sensitivity test of M.suis,rabbit HM,canine HMAfter infected RBCs of HMs were incubated with medium containing active drug,HM became free from IRBCs and move in medium,then movement became slow,parasites disappeared gradually.The PPE and parasites number on RBC decreased,movement of RBCs disappeared and their shape recovered.M.suis,rabbit HM,canine HM,were maintained in PPLO-S-BS containing 8 antibiotics respectively,the results showed that doxycycline hydrochloride was the optimal pesticide,PPE and PHE of these animal HMs decreased notablely due to the effecting of doxycycline hydrochloride or oxytetracycline hydrochloride(p＜0.05).The PHE and infected strength of IRBCS reduced markedly in medium containing tylosin tartaric(p＜0.05), but their PPE of these animal HM did not changed markedly.更多还原