| Object1. To Explore a simple and rapid method for identification of Sporothrix schenckii ,2. To know genomic character of Sporothrix schenckii ,3. To genotype 56 strains of Sporothrix schenckii ,4. To know the relationship between genotyping of Sporothrix schenckii and clinical menifestations of Sporotrichosis,5. To explore the relationship among virulence of 5. schenckii with genomic difference and clinical types of Sporotrichosis.Methods1. strains1. 1 56 isolates of Sporothrix schenckii , which were collected from different clinical types and various regions,1.29 other important clinical species including Aspergillus fumigatus , Exophiala jeanselmei , Phialophora verrucosa ,Cladosporium carrionii , Cryptococcas neoformans , Candida krusei , Candida albicans , Microsporum gypseum and Penicillium marneffei.2. animals108 male, 8 - week - old BALB/c mice ( average weight 22 ~ 24g) pur-chased from Animal Labortory Center of China Medical University were divided into two large groups, each of which was divided into 9 subgroups. Mice of the same age and body weight were used in one experiment. One experimental subgroup consisted of six mice. Groups of mice inoculated with the same strains of fungus were kept in one cage.3. DNA extractionThe genomic DNA was extracted from 56 strains of Sporothrix schenckii and 9 other important clinical species4. PCR-RFLP4.1 Amplify genomic DNA of 28 strains of Sporothrix schenckii and the other 9 important clinical species with the universal primers ITS1 and ITS4. 4.1.1 PCR reaction mixture contain; 50μl total volume template DNA (100ng/μl) 1μlMgCl2 (25 mmol/μl) 3μl,each of dATP, dTTP, dCTP, dGTP (2.5 mmol/μl) 1 μl mol,ITS1,TS4(20μmol/μl) 1μl,TaKaRa Taq (1. 25 U/μl) 1μl ,10 x buffer (free mg2+) 5μl4.1. 2 amplification condition(1) predenation 5min at 95 °C(2) 40 cycles of following procedure; 1 min at 951 ( denation) ,lmin at 681 ( annealing) ,1 min at 72°C ( extention).③ full extention 5 min at 72°C 4. 2 Digest the amplified products with restriction enzyme Hae III.4. 3 Digested products were electrophored in 2% agarose gels5. RAPDAmplify genomic DNA from 56 strains of Sporothrix schenckii with the a pair of random primers OPBG -01 andOPBG -14,5. 1. PCR reaction mixture contain :total volum 25μlOPBG - 01 (100 pmol/μl) 1μlSequence 5' - gtggctctcc - 3OPBG -14 (100 pmol/jjj) l|ilSequence 5 ' - gaccagccca - 3'OPBG - 19 (100 pmol/ui) 1 julISequence 5 ' - ggtctcgctc - 3 'OPB07(100 pmol/ul) lfxlSequence 5 ' - ggtgacgcag - 3 'Template DNA (lOOng/ u,l) 1 |xldNTP mixture( dATP,dTTP, dCTP, dGTP100|xmol) 4|xi10 x buffer ( contain mg2 + ) 2. 5jxlTaKaRaTaq (1.25U/|xl) 1 |xl5. 2 amplification condition: (D predenation 5min at 94 °C(2) 45 cycles of following procedure: 1 min at 94 Tl ( denation) ,lmin at 351 ( annealing) , 1 min at 12X1 ( extention).(3) A final 7 - cycle at 72°C ensured full extention of all of amplified products6. Experiment infection.6. 1 suspension preparation different concentration (4 x 10 cfu/ml, 1. 5 x 10 cfu/ml) of 5. schenckii suspension from disseminated sporotrichosis , regional lymphangitic sporotrichosis and regional lymphangitic sporotrichosis were made6. 2 Inculation6. 2.1 Footpad injection 0. 05ml( containing 20cfu) of 5. schenckii suspension from different manifestations of sporotrichosis was injected into the hind footpads of mice using a 27 - gauge needle and 1 - ml syringe(J)In Goup one, 0.05ml of S. schenckii suspension from disseminated sporotrichosis was injected into the hind footpads of miced) In Group two, 0. 05ml suspension of yeast form from regional lymphangitic sporotrichosis was injected into the hind footpads of mice;(3) In Group three, 0. 05ml suspension of yeast form from fixed cutaneous sporotrichosis was injected into the hind footpads of mice.6.2.2 Caudal Vein injection 0. 2ml of suspension ( containing 3 x 10 cfu) from different manifestations of sporotrichosis was injected into caudal vein(Din Group four, 0. 2ml of suspension( containing 3 x 106cfu) from disseminated sporotrichosisfrom DmuD, JD and CZ9784 was injected into caudal vein,(2)In Group five, 0. 2ml of S. schenckii suspension from regional lym-phangitic sporotrichosis was injected into caudal vein,(3)In Group six, 0.2ml of S. schenckii suspension from fixed cutaneous sporotrichosis was injected into caudal vein.6. 2. 3 Intraperitoneal injection mice was injected intraperitonealy with 0. 2ml of suspension ( containing 3 x 106cfu) from different manifestations of sporotrichosis(1) In Group seven, mice was injected with 0. 2ml of suspension( containing 3 xl06cfu) from disseminated sporotrichosisfrom DmuD, JD and CZ9784;(2) In Group eight, mice was injected with 0. 2ml of 5. schenckii suspension from regional lymphangitic sporotrichosis;(3) In Group nine, mice was injected with 0. 2ml of 5. schenckii suspension fixed cutaneous sporotrichosis.6. 3 Determination of CD4/CD8 in blood of mice inoculated with isolates of Sporothrix schenckii.6.4 Histopathological examination.6.4. 1 The swelling footpads, swelling skin on tails and visceral organs of mice inoculated respetively intraperitonealy, subcutaneously and intravenously inoculated mice were fixed in 10% formalin.6.4. 2 Thin (two - |xm) paraffin sections were examined by light microscopy after staining with PAS or H&EResults—. PCR-RFLP1. A 350bp fragment was amplified from all the 28 isolates of Sporothrixschenckii with the universal primers ITS1 and ITS42. The 350bp fragment was amplified from the other 9 important clinical species, among which them Exophiala jeanselmei , Cladosporium carrionii , Cryptococcus neoformans and Microsporum gypseum have another fragment of 53Obp3. An identical PCR - RFLP pattern from all the 28 strains of Sporothrix schenckii was different from that of the other 9 important clinical species~. RAPD1. The pattern of amplified products of Sporothrix schenckii from different regions were distinct with the two primers OPBG -01 and OPBG -14.2. The pattern of amplified products of Sporothrix schenckii from different clinical manifestation of Sporotrichosis with the two primers OPBG -01 andOP-BG-14;2. 1 The pattern of amplified products of Sporothrix schenckii from disseminated Sporotrichosis were was 1800bp, 1400bp, 800bp, 700bp and 520bp , which was different from regional lymphangitic sporotrichosis2. 2 The pattern of amplified products of Sporothrix schenckii from regional lymphangitic sporotrichosis were was 1400bp, 800bp, 700bp and 520bp, which was different from fixed cutaneous sporotrichosis2. 3 The pattern of amplified products of Sporothrix schenckii from fixed cutaneous sporotrichosis was 1800bp,800bp,700bp and 520bp.3. Acording to the pattern, 56 strains of Sporothrix schenckii from different regions and different clinical types were divided into 13 genotyping.IE. Different manifestations of mice after inoculation1. Earlier onset of illness and wider location of lesions and higher mortality rates of BALB/c mice inoculated by suspension of isolates from disseminated sporotrichosis were than by isolates from regional lymphangitic sporotrichosis2. Earlier onset of illness and wider location of lesions of mice inoculated by suspension of isolates from regional lymphangitic sporotrichosis were than by isolates from the fixed cutaneous sporotrichosis.3. For same a suspension of isolates from 5. schenckii , earlier onset of illness and wider location of lesions of mice inoculated by intravenous injection...
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