Research On The Cultrue And Pathogenesis Of Mycobacterium Marinum

Mycobacterium marinum (M.marinum)is a pythogenesis and atypicalMycobacterium,which is distributed all over the world.It resides in salt water andfresh water.It can infect fish and human beings by contacting.With the developmentand utilization of ocean,the disease incidence of co-suffering diseases shared byhuman and fish has a latent risk.At present,there were many reports aboutMycobacterium marinum infected human.The infecting incidence increasesobviously for mariculture staff,catching fish staff,fishmen and beautiful fishescultivation staff,etc.We have isolated M.marinum from pathological tissue,whichwas cultivated and drug sensitivity tested,and infected zebra fish and experimentmice.And we also have isolated pathological proteins from it and carried outpathopoiesis research.(1)Isolated experiment for Mycobacterium.We have isolated two Mycobacteriathat they were come from human'infected abscessus tissue(R1)and intravital bodysurface of crab (Y10),respectively.We have studyed them compare with typicalMycobacterium marinum in light reaction of cultivated colony,namely bring aboutyellow colony,and in drug sensitive test for nine drugs,and in acid-fast staining andchemical reaction,as well as the molecular weight of the strain wall-held protein.Wefound that they have consistency in above aspects,which can explain that they havecommon genes origin.(2)Zebra fish infectional tests:60 healthy immature zibra fish were chose,select30 at random,which were divided into three groups:A,B and C group.Every grouphas ten zebra fish.0.1 bacteria liquid which come from Y10,R1 and Mycobacteriummarinum were injected in intraperitoneal and cultivation in aquarium tank at 28℃respectively,to observe the living subsistence of zebra fish.Ten zebra fishes in trial group were died one after another in seven days by intraperitoneal injection 0.1mLMycobacterium marinum liquid.Pathological dim-Brownish red color tissue can beobserved in the dead zebra fish corpses when opened the abdominal cavity.Pathogenic strains of Mycobacterium marinum were isolated and cultivated frompathological tissues.(3)The infection modeling of mice and rates:12 healthy mice and rats werechosen respectively,divided into trial group and control group.According to thedifferent route of infection,the trial groups were divided into group A,B and C.Group A:trial mouse were given Mycobacterium liquid by vena caudalis intravenousinjection.Group B:trial mouse were given Mycobacterium liquid by intraperitonealinjection.Group C:trial mouse were given Mycobacterium liquid by alimentaryinfection.Control group mouse were given the same dosage normal sodium (NS)byvena caudalis intravenous injection.After one week infected,the trila mouse wereinfected again.Post-infected eight four weeks,venous blood from all the mouse venacaudalis were detected for cytokines (interleukin,interferon-gamma,tumour necrosisfactor-alpha),immunoglobulin(IgA,IgG and IgM),C-reaction protein andcomplements (C3 and C4)respectively.Cytokines was detected by enzyme linkedimmunosorbent assay(ELISA).Immunoglobulin,C-reaction proteiand andcomplements were assayed by automatic biochemistry analyzer.Mycobacterium marinum has pathogenicity to Kunming mice.Granuloma wasseen in the corpse by autopsy.The granulo-material was involved in granuloma,which was cheesy necrosis.The results of interleukin (IL-2,IL-4,IL-10 and IL-12)were no difference between trial A and trial B group after infected two weeks(p＞0.05),but higher than that of group C and control group(p＜0.05).IL-4 and IL-10 were moreincreased compared with IL-2 and IL-12(p＜0.05).There were statistical significancein IL-4 and IL-10 in group A and B,C andcontrol group(p＜0.01).The cytokine(sIL-2R and IFN-γ)from peripheral vein blood of infected eightweeks bigger rat were more enhanced in trial group A,B and C compared withcontrol group(p＜0.05).But there was no statistical difference in trial group A,B andC (p＞0.05).TNF-alpha was less increased secretion,but no difference between trial group A,B and C.The complement (C3 and C4)and C-reaction protein in trial groupA,B and C were no difference (p＞0.05).But there was statistical significancebetween trial group and control group(p＜0.05).The immunoglobulin (IgA,IgG andIgM)were no difference (p＞0.05)in trial groups A,B and C.While there wasstatistical significance between trial group and control group(p＜0.05).(4)we have drown off the tropina from Mycobacterium marinum,electrophoreticseparation,and carried out analysis.We have isolated 65kD proteantigen,which wascapital pathogenicity protein.490 protein spots were detected by two-dimensionalelectrophoresis,which molecular mass range from 10 to 100kD.The PI value ofprotein were mostly in pH 4.5 6.0,which were pH 4.5--5.5 relatively intensive.While the protein spots were rather scatter in pH5.5—6.0,namely the protein spotswere mainly in meta acids side,nothing were in alkalinity side.There is identity inpathogenity protein for the two isolated Mycobacteria and Mycobacteriun marinum.