Isolation Of Cryptococcus Neoformans From Pigeon Droppings And Analysis Of Its Serotype And Mating Type In Part Of Yunnan Province

Backgroud: Cryptococcus neoformans was recognized as a natural foci of conditions pathogenic fungi in 1894. C. neoformans could be detected from countryside farms, Bat Cave, rotting vegetables, fruits, insects, and healthy huma n mouth, surface, intestinal, pigeon droppings and other matrices. the positive rate and biomass of C. neoformans in pigeon droppings of all matrices are the highest. With chemotherapy drugs and corticosteroids were widely used, catheterization, peritoneal dialysis, organ transplants and others were popularly carried, especially in recent years, with the increase in human immunodeficiency virus(HIV) infection, the number of fungal infections has become the focus of public attention, the infection of C. neoformans was prominent particularly, mainly affects central nervous system and respiratory system of the human, leading to fatal meningoencephalitis and respiratory diseases, now C. neoformans were attracted attention of scholars widely. In China, the research of C. neoformans was focus on clinical, less research on the environmental strain of C. neoformans. In 2013, Linlina had studied 15 regions in China, and isolated and Identified 80 strains of C. neoformans in the environment, and found that clinical isolates of C. neoformans and environmental strains were high homology in China, so in order to avoid infection of C. neoformans, susceptible populations should not be in contact with the breeding environment of C. neoformans.Objective: The systems research of C. neoformans in different geographical distribution in Yunnan had not be carried yet, pigeon droppings was one of breeding environments for C. neoformans, The purpose of this study was to isolate and identify C. neoformans in old pigeon droppings and understand the distribution of its phenotypic and genotypic characteristics of the C. neoformans of environment strains in part of Yunnan Province. And could provide the reference for prevention, treatment and research of cryptococcal disease.Methods:1. Study Object: This study used Black Rice Agar Medium to isolate and identify 473 pigeon droppings from Dali, Zhaotong, Qujing, Lincang area in Yunnan.2. Experimental method:2.1 Comparison of caffeic acid cornmeal agar medium with black rice agar medium to selectively isolate C. neoformans: use the caffeic acid cornmeal agar medium and black rice agar medium to isolate and identify C. neoformans in pigeon droppings and to compare the isolating efficacy of caffeic acid cornmeal agar medium and black rice agar medium for C. neoformans in pigeon droppings.2.2 Isolation and identification of C. neoformans from pigeon droppings of part of Yunnan Province: This study used black rice agar medium to isolate C. neoformans from 473 pigeon droppings from Dali, Zhaotong, Qujing, Lincang area, and URA5 gene was amplified using the specific primer CN4 and CN5, identify according to the size of the amplified product.2.3 Analysis of serotype and mating type for C. neoformans of part of Yunnan Province: confirmed the serotype and mating type by specific primer.Results:1.The C. neoformans colony in the black rice agar medium appears nankeen and wet, like the glue liquor. 23 strains of C. neoformans were separated and the relevance ratio was up to 32.86 percent. The C. neoformans colony in the CACA was smaller than the size of the colony on the black rice agar medium. The colony turned out to be brown and dry. 11 strains of C. neoformans were separated from the medium and the relevance ratio was up to 15.71 percent. The relevance ratio of the black rice agar culture medium was higher than the that of CACA and was statistically significant(P<0.05). The number of flat of filamentary fungi grown on the black rice agar medium was obviously less than those grown on the CACA and the degree of pollution was lower than CACA. Through the ink coloring, it presented to be round or ovate and thick capsule by observing under the optical microscope, and the object2. Total 152 C. neoformans were isolated in all 473 specimens, in our experiment, the isolated rate of C. neoformans was 32.13% significantly(χ2=41.87,P<0.01), the positive rate of isolation in Dali was 26.08%, the positive rate of isolation in Zhaotong was 30.05%, the positive rate of isolation in Qujing was 81.81%, the positive rate of isolation in Lincang was 37.03%.3. All the 152 strains are serotype A, α mating type.Conclusion:1. The separating effect of Black rice agar medium as a selective medium was better than CACA.2.The positive rate of C. neoformans was high, and the positive rate of isolation was 32.13% in pigeon droppings in Yunnan, and the carrier rate is different in different areas.3. All the 152 strains are serotype A, α mating type.