Study On In Vitro Cell Free Culture Of Livestock Haemomycoplasmas

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.