Experiment Research On The Morphology Of Schizophyllum Commune And Its Animal Laboratory Study On The Pathogenecity

Objective: Schizophyllum commune belongs to Eumycophyta, Basidiomycetes, Agaricales, Schizophyllaceae, Schizophyllum. It widely distributes in whole world. Schizophyllum commune is saprophytic, which is a common invader of rotten wood, usually it was reported to be involved in cases of allergic bronchopulmonary mycosis after inhaling its sporus, seldom it produces the infection of deep tissues, but nowadays, there are many reports with regard to the infection of deep tissues at abroad. We isolated the strain from a patient's sputum in the clinic service. It was identified as Schizophyllum commune by China Institute of Microbiology in Acdemt Science.In our research, the morphology characteristics of Schizophyllum commune on the five kinds of culture media were observed to identify more suitable medium for its growth, which can help clinical docters to establish diagnosis on the infection of Schizophyllum commune. Furthermore, mice in different immune states were inoculated Schizophyllum commune through intradermal,intraperitoneal and intravenous pathways respectively. The death rate of the mice was observed during 28 days, the main viscera of the death or sacrificed in various stages were examined by retro-fungal culture and histopathology. The infected degree under each immune states were confirmed by fungi residual burden of heart, liver, spleen and lung. All of study can helped to know more about its pathogenicity and the change of tissue forms.Methods:1 Sequence analysis of DNA Extract DNA of the isolation and amplify the domain D1/D2 of its 26SrDNA, determin its site of basic group through DNA automatic sequencer, then search for its isogenic sequence in the nucleotide sequence library GenBank/EMBL/DDBJ and its genetic relationship in genealogical tree.2 Morphology observation on media2.1 General colony observation on the five kinds of media Put the isolation under the ordinary temperature rewarm about three days and inoculate it respectively on the media of Sabouraud′s Agar﹙SDA﹚,Malt Extract Agar﹙MEA﹚,Patato Glucose Agar﹙PDA﹚,Corn Meal Agar﹙CMA﹚ and Czapek's Agar﹙CZA﹚,and culture at 37℃and 27℃for two weeks, then observe its development and measure the diameters of colnies everyday,fianlly draw growth curves.2.2 Slide culture Inoculate the strain on the microculture of the above five kinds of media at 27℃and 37℃for two weeks and observe the growth of hypha and spores everyday.2.3 Observation under light microscope Put some samples taken from the five kinds of media colonies on the glass slide, cover them with slip when dissolved in the solution of cotton blue stain, then observe it under light microscope.2.4 Observation under scanning electron microscope The samples taken from the medium of SDA were fixed in the solution of 4% glutaric dialdehyde and prepared through routine procedure and examined under scanning electron microscope.3 Physiology experiments3.1 Urease test Inoculate the strain on the Urea agar,and culture it at the ordinary temperature for one week,then observe the change of the medium color.3.2 Cycloheximide tolerance test Inoculate the strains on the SDA with cycloheximide and the one without cycloheximide,then culture them at 37℃and 27℃for two weeks. Observe the result.4 Animal laboratory study on the pathogenecit4.1 Establition of animal model suffered from systemic infection of Schizophyllum commune The preserved fungi were activated, then inoculated onto the Sabouraud's Agar,culturing in 37℃for one week,then made up fungl suspension solution with isotonic Na chloride. Then mice in differents immune states were inoculated through intradermal, intraperitoneal and intravenous pathways respectively.4.2 Observing the death rate Observed the death rate of each group in the four weeks after infected the fungi and write down the death number and date.4.3 Observing the infectious organs Dissected the sacrificed mice in various stages , took the heart , liver , spleen , lung and kidney under sterile conditions,and observed their changes of size,color,and so on.4.4 Fungal retro-culture Under sterile conditions,the infected tissues were grinded and subcultivated into test tubes containing Sabourauds ager. After cultured at 37℃for one weeks,we counted the positive numbers. The colonies were subcultivated onto the media of plate culture and microculture. We observed the colonial morphology and shape of hyphae,spores.4.5 Observation of fungi residual burden of organs The heart,liver,spleen, lung and kidney of mice that inoculated Schizophyllum commune were homogenated under sterile conditions, then cultured on platiculture. The fungi residual burden of every organ was calculated.4.6 Histopathological Examination The organs of mice that inoculated Schizophyllum commune were removed out , fixed by 10 % Formalin, dehydrated , cleared , embedded , sliced , stained with HE and PAS respectively, then observed under light microscope.Results: 1 Results of DNA sequence analysis Based on the sequence analysis of 26S rDNA D1/D2 and the genealogical tree by Neighbor-Joining drawing , the rDNA domain of the stain was 100℅ consistency with Schizophyllum commune .2 General colonies observation on the five kinds of media The fungus growed faster in the first week on the SDA,PDA and MEA than CMA and CZA.The growth was better at 37℃than 27℃. All of the colonies were white,and woolly in appearance. 3 Draw growth curves The fungus growed faster in the first week on the five media , the comparison among the colony diameters on the five media showed that the colony diameters had no difference under the same temperatures(P>0.05).The comparison under the two temperatures on the same medium showed that the colony diameters at 37℃was larger than 27℃in the two weeks(P <0.05).4 Morphology under light microscope It showed abundant branched hyphae with septa. The mycelium were of rather diverse structure, with clamp connections and curved lateral pegs﹙spicules﹚.Medusoid fruit bodies and kidney-shaped conidiospore could be seen on SDA.5 Morphology under scanning electron microscopy Plentiful branched hyphae with septa, of rather diverse structure , often with clamp connections, short and curved lateral pegs﹙spicules﹚ and waterdrop-shape excretion. Conidia absent.6 Physiology experiments The test of urease reaction was positive ; Cycloheximide tolerance test was negative.7 The survival state of mice In the normal immune state,the life habits had no obvious change after inoculated with Schizophyllum commune. The mice that inoculated fungi through intradermal pathway had a nodue on the back. The immunosuppressed mice that inoculated intraperitoneally and intravenously reduced food-intake obviously, and emaciated gradually.8 The death condition of mice The immunosuppressed intravenously injected group reached its death peak in the third day after inoculation,whose death rate was 85℅,The intraperitoneally injected group began to die from the third day to the fourteenth day after inoculation,the death peak was at the sixth day,its death rate was 35℅,but other groups had no natural death. Comparing the immunsuppressed group inoculated intraperitoneally with the group inoculated intravenously, the death rate and mean survival time of mice had significant difference(P <0.05).9 The Change of Organs Parts of the intraperitoneal and intravenous injected mice in the immunsuppressed state had white microabscess in organs'surface,especially on the surface of liver,the number of which were more in the immunsuppressed intraperitoneal group. Other groups had no obviously changes.10 Fungal retro-culture When visceras were cultured at 37℃for three days, the fungi began to grow on part of viscera. The colonial morphology and shape of hyphae were identical with that of inoculated strains. In flat plate, the colonies presented white woolly type.The fungal retro-culture rate of the immunosuppressed group that inoculated intraperitoneally was obviously lower than the immunosuppressed groups that inoculated intravenously(P <0.05).11 Fungal burden of organs In immunosuppressed or normal state, The fungal residual burden of liver and spleen were more than other organs in the intraperitoneally inoculated group(P<0.05).The fungal residual burden of liver was more than other organs in the intravenously inoculated group(P<0.05).12 Fungal burden of livers In immunosuppressed or normal state, the infected degree of the immunosuppressed intravenously inoculated group were more serious than the immunosuppressed intraperitoneally injected group (P<0.05). Among immunosuppressed the intraperitoneally injected group and the normal groups that inoculated intraperitoneally and intravenously, the infected degree had no significant difference (P>0.05).13 Histopathological Examination when stained with HE,in the early stage, the important change was acute inflammation, with degeneration and necrosis of tissue cells. Lately , reactive hyperplasia of histocytes and polykaryocytes at periphery. fibrous encapsulation were also can be seen. Stained with PAS, violet branched hyphae could be found.Conclusion:1 Schizophyllum commune only had a colonial morphology of hyphomycete,it grows faster and better on SDA,which shows that the medium are the more suitable one for Schizophyllum commune. The strain grows better at 37℃than 27℃. Urease test is position,and the growth of Schizophyllum commune can be inhibted by cycloheximide. 2 Systemic infected murine models caused by Schizophyllum commune could be successfully established through intraperitoneal and intravenous pathway in this study,but not intradermal.3 The histopathological change of infected tissues was acute or chronic nonspecific inflammation , with fibrous encapsulation forming. Mice in immunosuppressed state were easier to be infected,whose degree were more serious. Health mice also could be infected disseminatedly when inoculated more fungi. Schizophyllum commune is a opportunistic fungus,its virulence was correlative with immune state.4 Liver was the most common involved organs.