Sequence Analysis Of The ITS Of Rabbit Fungal Dermatopathy Of Shandong

Dermatophytes have been classified into three genera, Trichophyton, Microsporum and Epidermophyton. They invade keratinized tissues of human and other animal, that is, superficial cornified skin layers, hair and nails, causing asuperficial cutaneous infection called dermatophytosis. Dermatophytes which produced an infection to laboratoryanimal are mainly Trichophyton mentagrophytes, Microsporumgypseum and Microsporum canis. The three kinds of dermatophytes should be banned to laborary animal, no matter they were isolated in infectious sites or uninfectious sites. Because the dermatophytes in uninfectious sites was probable causing dermaophytosis of laboratory animal and producing infection to laboratory man.China has become the biggest rabbit raising country in the world, especially ShanDong Province, and dermatophytosis of the rabbit has caused great losses to it. The current diagnosis of dermatophytosis is based upon the status of colony and microscopic identification of hyphae directly from lesion materials followed by in-vitro culture. Although rapid and economical, microscopical examination is not species-specific and needing a lots of experience.In fact,the phenotypic characteristics of dermatophytes can be easily influenced by outside factors,such as temperature variations,chemotherapy and culture media,that may interfere with the metabolic process of the dermatophyte and affect the interpretation of in-vitro culture results.In-vitro culture is capable of providing typical characteristics of morphological and biochemistry in 10~15 days. However,for some unusual and atypical characteristic dermatophytes,identification may require a range of special culture media. These tests will consum several weeks. Therefore, establishing the dermatomycosis's rapid diagnostic methods, isolated and acquired rabbit dermatophyte pathopoiesis bacterial strain in Shandong province , verifying its variation situation and tolerance , it is important to prevent and cure this disease . On the basis of comparing the advantage and disadvantage of molecular biology methods and the conventional laboratory testing methods to identify dermatophyte pathogeny, Shandong popular bacterial strain was isolated and acquired. The isolated strains of the variation and genetic evolution relationship was discussed through cloning, sequencing to dermatophyte ITS gene, and then we acquired the satisfactory results by doing the tolerance detection of isolated strain. This research contains three parts:Firstly, design of universal primers based on specific rDNA sequences of dermatophytes can detect the known bacterial strain and other microorganisms by PCR. The PCR products are cloned and sequenced. PCR technology which is high sensitivity, speciality and rapidity has been used for dermatophytes identification. The results reveal that the detected and known bacterial strains were amplified a specific sequence of 650bp-770bp, but the other microorganisms, rabbit somatic cells and health rabbits were not. In addition, the detectablerate of PCR is higher than the method of conventional isolation and culture identification(82.8% and 60.55%). Then nine strains of rabbit dermatophyte were isolated and identified from major rabbit-farm in Shandong province, concluding 6 strains of Trichophyton mentagrophytes, 2 strains of Microsporum gypseum and 1 strian of Microsporum canis.Secondly, universal primers are designed according to conserved sequence internal transcribed spacers of dermatophytes, 16 strains of dermatophyte collected from major rabbit-farm in Shandong province were cloned and sequenced on ITS area. The result displays that the sequence of 5.8S rDNA in different pathogenic bacteria is highly conserved, but the variability of ITS is higher. Alignment analysis indicated that the length and the base composition of ITS1 were more variable than that of ITS2 among the species. Whereas in any species no difference was shown in the length of ITS1 and ITS2. The base composition was shown faintly diverse, so, rabbit dermatophyte were identified based on this area. This study confirmed that target sequence of PCR detected special primer by rabbit dermatomycosis pathogenic. It provides reliable drone for the special molecule identification of rabbit dermatomycosis pathogenic, and provides basis of molecule for scientific classes of rabbit dermatiphyte. At the same time, the different result from traditional morphology class was got by cloned, sequenced analysis ITS of isolated strain. Traditional morphology think: Microsporum gypseum and Microsporum canis are Microsporum, Trichophyton mentagrophytes and Trichophyton rubrum are Trichophyton; But this experiment sequence analysis results show that the homology of Microsporum gypseum and Trichophyton mentagrophytes is closer than the homology of Microsporum gypseum and Microsporum canis. The final conclusion still need further discussion.Finally, the resistance of rabbit dermatophyte pathogenic in Shandong province is analyzed by using ATB FUNGUS 3 to do drug sensitive test. The result reveals that isolated dermatophyte pathogenic bacteria is sensitive to Amphotericin B, 5-Fluorocytosine, Fluconazole , Itraconazole and Fconazole, respectively 83.33%,68.75%,75% and 64.41%. And Fluconazole is only with resistance 20.83%. Microsporum canis is most sensitive to Amphotericin B (87.5%), Trichophyton mentagrophytes and Microsporum gypseum are lower(81.25%). The test results have provided scientific basis for scientific prevention of rabbit dermatomycosis.