Identification And Pathogenicity Of Aspergillus Nidulans From Goat

Objective: In order to identify the strain XM06 from goat and to investigate its pathogenic molecular mechanism.Methods: The potato dextrose agar medium(PDA)culture,lactophenol cotton blue staining,scanning electron microscopy and ITS gene amplification were used for the isolation and identification of strain XM06.The pathogenicity and pathogenic mechanism of Aspergillus nidulans strain XM06 in mice were investigated by serum biochemical parameters,tissue fungal load,HE staining,Masson staining,TEM,RT-q PCR,immunofluorescence and transcriptome sequencing.Results: The strain XM06 was dark green on PDA with a white fluffy margin and a powdered centre.The strain XM06 had short conidia with smooth walls and spherical conidia with a rough surface.The ITS gene fragment of strain XM06 was 571 bp and was 99.8% homologous to Aspergillus nidulans strain PWQ2388(KP131594).The strain XM06 was identified as A.nidulans.The A.nidulans strain XM06 carried Cat1,Rho1,Pka R,Alp2,Aspf1 virulence genes.The A.nidulans strain XM06 was resistant to amphotericin B and susceptible to itraconazole,caspofungin and voriconazole.The morbidity rate in the treated group of mice infected with Aspergillus nidulans strain XM06 was 66.7% and the mortality rate was 33.3%.Compared to the control group,the activities of serum alanine transaminase(ALT),glutathione transaminase(AST)and alkaline phosphatase(ALP)(P<0.01)and the concentrations of total protein(TP),globulin(GLO)and C-reactive protein(CRP)in the treated group were highly significantly increased(P<0.01),but the concentrations of albumin(ALB)and the ratio of albumin/globulin(A/G)in the treated group were highly significantly decreased(P<0.01).Compared with the control group,the body weight and average daily feed intake(ADFI)in the treated group were significantly decreased(P<0.05).Compared with the control group,the organ indices of the liver,spleen and brain in the treated group were highly significantly increased(P<0.01),the organ index of the lung in the treated group was significantly increased(P<0.05),but the organ index of the intestine significantly decreased(P<0.05).The fungal load of the spleen and liver were significantly increased than that of the other organs(P<0.05).Compared with the control group,the myocardial fibers were ruptured,lymphocytes and eosinophils were infiltrated in the liver,macrophages and lymphocytes were increased in the white pulp of the spleen,alveolar walls were thickened,renal epithelial cells were exfoliated,and the intestinal villi were broken,and the blood vessels in the brain were congested in the treated group.Compared with the control group,the collagen fibres inside the liver were hyperplastic,the structure of the hepatocyte cytoplasm was loosened,the mitochondrial cristae were reduced,the autophagic vesicles in the cytoplasm were increased,and the mitochondria in the splenic lymphocyte cytoplasm were swollen in the treated group.Compared with the control group,the m RNA expressions of LC3 A,LC3B and Beclin-1 in the treated group were significantly increased(P<0.05),but the m RNA expressions of m TOR and P62/Sqstm1 in the treated group were highly significantly decreased(P<0.01)and the fluorescence intensity of LC3 B in the treated group was highly significantly increased(P<0.01)in liver.Transcriptome sequencing results showed that 47.2 million and 44.9 million clean reads were obtained in the control group and treated group respectively.946 differentially expressed genes(DEGs)were obtained,including 372 up-regulated genes and 574 down-regulated genes.GO analysis showed that these DEGs were mainly involved in the immune responses,antigen binding,immunoglobulin receptor binding,bacterial defence responses,endopeptidase activity and so on.Moreover,KEGG analysis showed that these DEGs were mainly enriched in the following pathways: complement and coagulation cascades,retinol metabolism,cytochrome p450,IL-17 signalling pathway and so on.Additionally,RT-q PCR results showed that the trends and differential ploidy Vtn、Mbl2、Serpin D1、Cxcl2、Cxcl5、MMP9、MMP13、S100A and Fcgr3 genes were consistent with the RNA-Seq results.Conclusion: The goat-derived strain XM06 was identified as A.nidulans,which was highly pathogenic in mice,caused autophagic death of hepatocytes,and caused spleen inflammation and differential expression of immune-related genes.