| Sporothrix schenckii is the causative agent of sporotrichosis,which affects primarily the skin, subcutaneous tissue, lymph vessels, lymphnodes and rarely ,visceral organs. Although fixed lesions are common, disseminated infection by Sporothrix schenckii can be found occasionally. The clinical manifestations of sporotrichosis are related to immune status of the individual, and whether they are related to different strains is unknown. The conventional taxonomy of fungi has been mainly based on morphology. A problem of Sporothrix schenckii morphotyping is that S.schenckii can not be sub-divided within the species. In this study, we attempted to subtype S.schenckii species by a much straightforward method through restriction fragment length polymorphisms analysis of the ribosomal-DNA repeats .The relationship between DNA patterns and geographic origins and clinical manifestations of S.schenckii were also studied by this method. We also investigate the mycologic features and variations in gene sequence of a sporothrix strain (Dmu11) which caused disseminated cutaneous sporotrichosis. Materials and Methods: 1. Thirty-one clinical isolates of S.schenckii collected from different areas and isolated from patients with different clinical manifestations were included in this study. The total DNA was extracted by cetyltrimethyl ammonium bromide (CTAB). The polymorphisms were detected by hybridization of Apaâ… -digested S. schenckii genomic DNA with a probe amplified from the small-subunit rDNA and adjacent internal transcribed spacer (ITS) regions. The specific primers NS5[5'AACTT AAAGG AATTG ACGGA AG 3'] and ITS4[5'TCCTC CGCTT ATTGA TATGC3'] were adopted, and the probe was labeled by ECL kit . The band patterns manifested by southern blotting were used as the basis to study the genetic typing of 31 strains of S. schenckii collected from different areas (20 from Dalian, 5 from Shanghai, 3 from Beijing, 2 from Changchun and 1 from Nanjing). 2. The isolate Dmu11 was identified by routine mycologic and polymerase chain reaction(PCR) techniques. Ribosomal conserved sequence and non-transcribed spacer (NTS) were determined. Results: 1. The band patterns of genomic DNA from isolated strains after complete digestion by ApaI were smear-like. However, several clear and stable band patterns were obtained after hybridization with the probe by southern blotting.2. Fifteen individual patterns (DNA Type A-O) were recognized among the 31 isolates examined. Three patterns, type A, B and C accounted for 51.61% of all the strains .In all the 31 clinical strains in this study a universal band at 1.6Kb can be demonstrated. 3. The band patterns of isolates from southern China are markedly different from those from northern regions. Northern strains have 3-6 bands with the universal bands at 1.6Kb.The type A was most abundant, followed by type C , with only one isolate from type B. On the other hand, southern strains have 5-6 bands with the universal bands at 1.6Kb and are represented by type B, but none of types A and C. 4. There were significant differences in the hybridization profiles in isolated with different clinical manifestations. Type H DNA Pattern was represented by a single isolate from disseminated sporotrichosis. 5. The clinical isolate causing disseminated cutaneous sporotrichosis was identified as S. schenckii by routine morphological andPCR techniques. The sequence variation of NTS was found in the strain with a 10bp delection in the NTS segment as compared with the localized and lymphocutaneous strains. Conclusion: 1. This is the first time that Sporothrix schenckii strains were sub-divided within the species by the RFLP analysis of ribosomal DNA.2. The DNA typing of S. schenckii by southern blotting analysis of ribosomal-DNA intergenic spacer regions was highly sensitive and fifteen different DNA band types were recognized among the 31 isolates examined.3. We also found a obvious correlation between DNA patterns and different geographic areas and clinical manifestations.4. It seems that the technique of typing S.schenckii...
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