| Objective: Backusella belongs to the Eumycota,zygomycetes subphylum,Class Zygomycetes,Mucorales.It is widely distributed in the soil,excrement and rotting bodies of animals and plants.It is usually conditional pathogenic bacteria,which can lead to superficial mycoses or deep mycoses when the immunity is lower.B.circina var.circina is one variant of Backusella,but there is no related literature report of human infection with the fungus by far.We isolated the strain from one case of facial skin lesions on the patient who has got leukemia in 2009,and it was identified as Backusella circina var.circina by Institute of microbiology Chinese academy of sciences.Its gene sequence has been detected.This study could provide the basis for laboratory identification of this fungus by further morphological observation of B.circina var.circina on the SDA medium.On the other hand,the possibility of systemic infection could be detected by animal experiments.We observed the general condition and death situation of mice in different immune status and inoculation ways.The main viscera in various stages were examined by fungal culture and histopathology.All of study could help us to know more about its pathogenicity and pathological features,further more,it could provide powerful basis for clinical diagnosis and treatment on the infection of B.circina var.circina.Methods:1 Morphology observation on the SDA medium1.1 Colony general morphology observation: Put the isolate under the ordinary temperature about three days and inoculated it on the SDA medium,cultured it under 27? and 37?,then observed the development situation everyday.1.2 Slide culture: Inoculated the strain on the microculture of SDA medium at 27? and 37?,and observed the growth of hyphae and spores everyday.1.3 Observation under light microscope: Put some samples taken from the SDA medium colony on the glass slide,added a few drops of lactophenol cotton blue and took a coverslip on them.Then observed it under light microscope.2 Animal pathogenic experiment2.1 Establishing animal model with systemic infection of B.circina var.circina: Put the isolate under the ordinary temperature and inoculated it on the SDA medium at 27? for 3 days.Then made up the fungal suspension with sterile normal saline.Mice in differents immune states were inoculated the fungal suspension through intradermal and intraperitoneal pathways.2.2 Observation of activity condition and death rate: Observed the death rate and activity condition of each group in the four weeks after infected the fungi,and recorded the date and number of death.2.3 General observation of the viscera: Dissected the sacrificed mice in various stages under the aseptic conditions,and observed visceral changes of size,color,and so on.2.4 Fungal culture of the tissue: Grinded the infected tissues of mice in sterile conditions,then inoculated some of them into test tubes which containing Sabourand's Agar.After cultured at 27? for 3 days,we counted the positive numbers,and observed the colony morphology and the shape of hyphae and spores.2.5 Histopathological examination: The lesion tissues were fixed with 10% formalin,then dehydrated,cleared,embedded,sliced,stained with HE and PAS respectively.Finally,observed under light microscope.Results:1 The colony diameters on the SDA medium: The colony growed faster at 27? than 37? on the SDA medium.There are significant differences on the first,third and seventh day(P<0.05).2 The morphology of colony on the SDA medium: We could see colony of white and woolly.The colony was sparse,fluffy and obviously upward growth trend at 27?.The edge of the colony was not neat at 37?.Medium on the back was white both at 27? and 37?.3 Morphology under light microscope: It showed abundant hyaline branched hyphae.The mycelium were of uneven diameter.A large number of sporangia gathered at the top of hyphae.Many circular spores were released when sporangia burst.4 The survival state of mice: No matter what kind of immune status,the life habits had no obvious change after inoculated through intradermal pathway.The skin lesion of mice that immunosuppressed were more serious.The mice that inoculated intraperitoneally reduced food-intake obviously and emaciated gradually,the immunosuppressive mice situation was more serious.5 The death condition of mice: The immunosuppressed intraperitonaelly injected group reached its death peak in the fifth day after inoculation,and its death rate of one week was 56.7%,and two weeks was 96.7%.The intraperitoneally injected group which has normal immune system reached its death peak in the ninth day,and its death rate of one week or two weeks was 30% or 80%,and after two weeks without a natural death.By comparing the above two groups,we found that the death rate and mean survival time of mice have significant difference(P<0.05).And other groups had no natural death.6 The change of organs: The organs of intraperitoneally injected mice could be found in different levels of congestion and edema.Parts of the liver,spleen and lung had white microabscess on the surface.Intradermal inoculated groups had no obviously changes.7 Fungal culture of the tissue: When the intradermal inoculation,the skin tissue fungal culture result of the immunosuppressed mice was positive,but the skin tissue fungal culture of the normal mice didn't see the colony growth,and the fungal culture results of viscera in immunosuppressed and normal groups were negative.When the intraperitoneal inoculation,the viscera tissue fungal culture could grow out colonies.The colonial morphology and shape of hyphae were identical with that of inoculated strains.The fungal culture positive rate of the immunosuppressed mice was significantly higher than the normal mice(P<0.05).The fungal culture positive rate of the spleen,lung and liver were significantly higher than the heart and kidney(P<0.005).8 Histopathological examination: Different immune state of intradermal inoculated mice didn't show obviously pathological changes of organs.When stained with HE,the important pathological changes of intraperitoneally inoculated mice were thrombosis and tissue necrosis,with a large number of inflammatory cells infiltration.Stained with PAS and HE,there were a lot of purple hyphae and spores in the necrotic tissue and blood vessel.Conclusions:1 There was only one colony morphology of B.circina var.circina.And the colony growed better at 27? than 37? on the SDA medium.2 Systemic infected murine models caused by B.circina var.circina could be successfully established through intraperitoneal pathway in this study,but not intradermal.3 Backusella circina var.circina is an opportunistic pathogen.Mice in immunosuppressed state were more susceptible to be infected,and the degree of infection was more serious also.When inoculated more fungi,healthy mice could be infected as well.Therefore,the virulence and invasiveness of this fungus is very powerful.4 The characteristic pathological changes of mice caused by this fungus were easy to invade blood vessels,and could lead to thrombosis and tissue necrosis,with a large number of inflammatory cells infiltration.5 The liver,spleen and lung were the most susceptible organs. |
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