| Objective: Scopulariopsis brevicaulis is a saprophytic conditional pathomycte, usually it causes tinea unguium, seldom it produces the infection of deep tissues, but now there are many reports with regard to the infection of deep tissues. We isolated the strain from a patient of tinea unguium in the clinic service. It was identified as Scopulariopsis brevicaulis by China Institute of Microbiology in Acdemt Science.In the research, The isolation was observed with full-scale for further understanding of characteristics on morphology and vegetative production of Scopulariopsis brevicaulis which can help clinical docters to establish diagnosis on the infection of Scopulariopsis brevicaulis. Further more, mice in different immune state were given Scopulariopsis brevicaulis, through differently injected pathway. The death of the mice was observed, the main viscera of the death or sacrificed in various stages were examined by fungal culture and histopathology. The infected degree of each organs were confirmed by Fungus burden of kidneys and stained histological slide. Electron microscope was used to observe the morphilogy of fungal cells in tissue. All of study can helped to know more about its pathogenicity and 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/PD and its genetic relationship in genealogical tree.2 The whole colony observation: Put the isolation under the ordinary temperature rewarm about 24 hours and inoculate it onto the medium of SDA, culture it under 37℃and 27℃, then observe its development everyday.3 Observation under light microscope: Put some samples taken from the type of mycelial and yeast-like colony on the glass slide, cover tham with slip when dissolved in the solution of 10%KOH, then observe it under light microscope.4 Observation under scanning: electron microscope The samples from the colonies were fixed in the solution of 4% glutaric dialdehyde and prepared through routine procedure and examined under scanning electron microscope and transmission electron microscope respectively.5 Establishing murine model of the systemic Scopulariopsis caused by Scopulariopsis brevicaulis.: The fungi preserved in our department were removed from the freezer and let warm, then inoculated into the Sabouraud's Agar, culturing in 27℃. Ten days later, making up the fungal suspension solution. Then mice in differents immune states were inoculated through intradermal, intraperitoneal and intravenous route.6 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.7 Observing the infectious organs: The main viscera of the dead or sacrificed mice in various stages were removed and observed their changes of size, color, and so on.8 Direct Microscopic Examination: removed a small portion of tissue and teared apart, then put on a microscope slide, adding one drop of KOH solution. Added a coverslip, heated gently and then pressed down on the coverslip. Examined microscopically, first at low power and then using the high dry lens.9 Tissue Culture: grinded the infectious tissue aseptically in a sterile mortar with a pestle, then pick up some and culture on medium: test tubes, After culturing the tissue in 27℃for three days, observed the colonies form, color and so on. Then seeked our some to examine microscopically.10 Histopathological Examination: pathological tissues of the white patches were removed out, fixing, dehydrating, clearing, embedding, slicing, stainning with HE and PAS respectively, then observed under light microscope.11 Fungal Burden of kidneys: grind kidneys in a sterile mortar and pestle ,adding one ml of saline, then remove out tenμl of tissue solution and culture on petri dishes mediums in 27 ℃.Two days later, read the colonies number.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 stain is closest to Scopulariopsis brevicaulis in genetic relationship .2 Morphology under naked eyes: When cultured for 3-4 days,they began to grow presented with whitish hymeno-type colony, growing fast , they became dust color.3 Morphology under light microscope: It showed abundant branched,separate mycelium and penicilli. conidiospore grew from annellophore with cluster ,globular, thick wall, slick or rough hard sphere.4 Morphology under scanning electron microscopy: It showed penicilli,and spherical conidium produced from the lateral surface or end of branched mycelium.5 Animal motel: Mice were inoculated through intraperitoneal and intravenous injection pathway, Fungal cultures and Histopathological examinations were also positive. Mice injected intradermally had no changes. Fungi cultures and Histopathological examinations of each organs showed negtive.6 Death Rate: The intravenously injected group reached its death peak in the third day after inoculation, whose death rate was 93.3%. The intraperitoneally injected group began to die from the third day to the fourteenth day after inoculation, the death peak was at the seventh day, its death rate was 53.3%.There was no natural death in intradermally inoculated group and all the controls.7 The Change of Organs: It showed splenohepatomegalia or atrophy with prunosus. And partial lungs atrophy with peach.8 Direct Microscopic Examination: There were a lot of round or oval spores as well as branched filaments.9 Tissue Culture: The colony was white hymeno-type ,later became beige at 27℃when cultured for three days.10 Histopathological Examination: In the early stage, the important change was acute inflammation, with necrosis of tissue cells. Lately, when stained with HE and PAS, hyphae and reactive hyperplasia of histocytes and polykaryocytes at periphery. fibrous encapsulation were also can be seen. Stained with PAS, violet and red hyphae and round spores could be found.11 Fungal Burden of kidneys: In immunosuppressed or normal state, the infected degree of the intravenously inoculated group were more serious than the intraperitoneally injected group(P<0.05). In the intraperitoneally injected group, the infected degree of the immunosuppressed were more serious than normal state(P < 0.05).In the inoculated intravenously group, the infected degree of the immunosuppressed were more serious than normal state(P<0.05).Conclutions:1 Scopulariopsis brevicaulis only had a colonial morphology of hyphomycete, it showed abundant branched,separate mycelium and penicilli. conidiospore grew from annellophore with cluster, globular, thick wall, slick or rough hard sphere. Surround marks gaves birth to spores .2 Systemic infected murine models caused by Scopulariopsis brevicaulis could be successfully established through intraperitoneal and intravenous pathway in this study, but not intradermal.3 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.4 The histopathological change of infected tissues was acute or chronic untypical inflammation, with fibrous encapsulation forming.5 The death rate and the fungal burden showed that the infected degree of mice injected intraperitoneally was more serious than intravenously.
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