Sequencing Analysis Of Gene Frame Map And Pathogenicity And Drug Resistance Of Candida Tropicalis In Dairy Cow Mastitis

Cow mastitis is one of the most important diseases in the global dairy industry.Among the many pathogenic factors of dairy cow mastitis,microbial infection is still the main cause.In addition to the common Gram-positive bacterial infections,fungal infections are on the rise day by day.Candida tropicalis is a conditioned pathogenic fungus,which has been widely studied in human diseases,but there are few reports on Candida tropicalis in dairy cow mastitis.Therefore,in this paper,the fungi were isolated and identified from the diseased cattle suffering from cow mastitis in a dairy farm in Guangxi,and the tropical Candida albicans were isolated,and then the genome frame map was sequenced.the sequencing results were analyzed by genome function analysis and comparative genome analysis,and combined with pathogenicity test and drug sensitivity test,drug resistance genes and pathogenicity were analyzed.The results are as follows:1.Sequencing of the genome frame map of Candida tropicalis.Through the sequencing of the isolated Candida tropicalis,a full-length 14.27 Mb genome was obtained.By annotating the gene sequence,we obtained 8164 coding genes,200 non-coding RNA with a total length of 18290 bp,including t RNA 166 bands,sn RNA 33 bands and mi RNA 1 bands.The average GC content was 32.97%.There are 3241 repeats,including 1255 long terminal repeats,1262 DNA transposons,38 short repeats and 51 RC.Comparing the sequencing results with the database,the results of gene functional annotation showed that there were at most 4068 genes related to Candida tropicalis in NR database,and 3921 genes were annotated in GO database,which were involved in biological process,cellular composition and molecular function.There were 125 carbohydrate enzymes annotated by CAZy;4673 genes were annotated in KEGG database,which were divided into 6 functional pathways and 11 metabolic pathways,of which 36 genes were involved in drug resistance pathway;2276 genes were annotated in KOG database,involving 25 functions.In addition,197 secreted proteins,14 cytochrome P450 proteins and 9 Candida tropicalis were predicted.Compared with PHI database,it was found that the chemical resistant mutation of gene cyp51 and the virulence-enhanced mutation of gene VTC4 were found.2.Comparative genomic analysis of Candida tropicalis.The Candida tropicalis strains isolated in this experiment were compared with the known reference sequences in the NCBI gene bank.The results showed that there were a large number of translocation regions between the sample strains and the reference strains,indicating that there were some structural differences between the two genomes.SNP and In Del analysis showed that there were 30683 SNPs between the two genomes and 6644 In Del mutations in the coding region of the gene,including 3166 insertions and 3478 deletions.Compared with the reference strain,there are 2576 specific genes in the sample strain.There are SNP non-synonymous mutation and In Del non-synonymous mutation in the coding region of CDR1,CDR2,CDR3,CDR4 and ABC protein families of Candida tropicalis.These non-synonymous mutations can change the types of amino acids and may affect the expression of drug resistance proteins.3.Pathogenicity and drug sensitivity test of Candida tropicalis.The pathogenicity test was carried out by intraperitoneal injection of Candida tropicalis suspension in mice.the results showed that Candida tropicalis could damage the tissues and internal organs of mice,resulting in different degrees of cyan in the abdominal cavity of mice.there are white necrotic foci on the surface of visceral organs and pulmonary bleeding.The spleen and thymus are swollen.According to the observation of pathological section,there were obvious vacuolar degeneration in hepatocytes,blurred boundaries between cells and stenosis of hepatic sinusoids.Alveolar septum thickening,pulmonary venule and alveolar septum telangiectasia,and filled with a large number of red blood cells.The medulla and cortex of the thymus are blurred and there are a large number of red blood cells.The structure of renal tubules is disordered and the capillaries are diffusely congested.The drug sensitivity of the sample strains to drugs was detected by drug sensitivity disk diffusion method and constant drug gradient method.The results of drug sensitive disk diffusion method showed that the sample strains were highly sensitive to nystatin and ketoconazole,moderately sensitive to amphotericin B,and insensitive to itraconazole,gentamicin,penicillin and ciprofloxacin.The sample strains were sensitive to the gradient changes of ketoconazole,but insensitive to the concentration gradients of fluconazole,itraconazole and amphotericin B.Conclusion: there is Candida tropicalis infection in dairy cows in Guangxi,and the isolated strains are pathogenic and resistant to itraconazole and fluconazole.After functional annotation and reference genome comparison,the drug resistance genes of Candida tropicalis isolated in this experiment have mutations and may evolve.