微小根毛霉对小鼠的致病性研究

Study on pathogenicity of Rhizomucor pusillus in mice

为探究微小根毛霉的致病性,本研究将微小根毛霉孢子悬液以6×107cfu/mL剂量感染昆明小鼠,观察小鼠临床症状和剖检病变,结果显示:感染小鼠出现精神萎靡、弓背、站立不稳等症状,死亡率为20%;剖检发现心肝脾肾肿大、肝脏表面有黄白色结节、肺脏多处出血灶。小鼠感染7 d后眼眶采血分离血清,检测血清生化指标,结果显示:感染小鼠血清中的谷草转氨酶(AST)和碱性磷酸酶(ALP)含量均显著升高(p<0.01),谷丙转氨酶(ALT)含量显著下降(p<0.01),总蛋白(TP)和球蛋白(GLB)含量显著升高(p<0.05),提示感染小鼠肝脏功能受到损伤;尿素(UREA)、尿酸(UA)和肌酐(CREA)含量均显著性增加(p<0.05),提示感染小鼠肾脏生理功能受到损伤。同时剖杀小鼠,制作各脏器的病理切片,光学显微镜下观察,结果显示:感染小鼠心肌中有霉菌侵入,心肌纤维变性;肝细胞肿胀、变性,汇管区有大量炎性细胞聚集;脾组织中可见菌丝和大量的孢子,中性粒细胞增多、淋巴细胞减少;肺泡膈充血、炎性细胞浸润,支气管上皮细胞脱落;肾组织发现少量菌丝,大量肾小管上皮细胞变性、脱落和坏死,肾小球萎缩;小肠组织发现霉菌孢子,黏膜固有层有淋巴细胞浸润。进一步利用透射电镜观察小鼠的肝脏和肾脏的超微组织病理学变化,结果显示:感染小鼠肝细胞肿胀,染色质稀少,线粒体增生、肿胀,胞浆电子密度降低;肾小管上皮细胞体积变小,胞核碎裂,线粒体高度肿胀,嵴断裂且模糊不清,胞浆多处空泡。采用TUNEL染色法染色感染小鼠肝肾组织后于荧光显微镜下观察,结果显示:实验组小鼠大量肝细胞和肾细胞均发生了凋亡。综上所述,微小根毛霉对小鼠具有一定的致病性。本研究首次为阐明微小根毛霉的致病机制提供系统的组织病理学资料。

In order to explore the pathogenicity of Rhizomucor pusillus,Kunming mice were infected with the spores suspension of Rhizomucor pusillus at a dose of 6×107 cfu/mL.The results showed that the infected mice had symptoms such as listlessness,arched back,and unstable standing,and the mortality rate was 20%.Clinical anatomies showed enlargement of the heart,liver,spleen and kidneys,yellow-white nodules on the liver surface,and multiple hemorrhages in the lungs.The serum biochemical indexes of infected mice were detected at 7 days after infection.The results showed that the contents of aspartate aminotransferase(AST)and alkaline phosphatase(ALP)were increased(p<0.01)in the serum of infected mice,while the content of alanine aminotransferase(ALT)was significantly decreased(p<0.01),and the contents of total protein(TP)and globulin(GLB)were increased(p<0.05),indicating that the liver function of infected mice was damaged.The concentrations of urea(UREA),uric acid(UA)and creatinine(CREA)were significantly increased(p<0.05),suggesting that the physiological function of the kidneys of infected mice was impaired.The mice were dissected at 7 days after infection,and the histopathological changes of the heart,liver,spleen and other parenchymal was observed under optical microscope.The results revealed that myocardial tissue of infected mice had mold invasion and myocardium fiber were denatured;the hepatocytes were swollen and degenerated,and a large number of inflammatory cells gathered in the portal area.The spleen tissues were invaded by mycelium and a large number of round spores,with neutrophilia and lymphopenia.The alveolar septum was congested and infiltrated by inflammatory cells,and bronchial epithelial cells were exfoliated.A small number of mycelia were observed in the renal tissues,a large number of renal tubular epithelial cells were degenerated,exfoliated and necrotic,and the glomeruli were atrophic.Many mold spores were found in the small intestine,and lymphocytes infiltrated the lamina propria of the mucosa.The ultrastructural changes of liver and kidney of infected mice were further observed by transmission electron microscopy.The results showed that the hepatocytes of the infected mice were swollen,chromatin was scarce,mitochondria were proliferated and swollen,and the cytoplasmic electron density was reduced.The epithelial cells of renal tubular were reduced in volume,the nuclei were fragmented,the mitochondria were highly swollen,the cristae were broken and blurred,and many vacuoles exist in the cytoplasm.The liver and kidney tissues of infected mice were observed by TUNEL staining,and the results showed that apoptosis occurred in a large number of hepatocytes and renal cells in the infected mice.In summary,Rhizomucor pusillus can cause certain pathogenicity to mice.This study provides valuable histomorphological data for the first time to elucidate the pathogenic mechanism of Rhizomucor pusillus.