Isolation Of Avian Aspergillus Fumigatus And Development Of Quantitative PCR Method For Detection

Aspengillosis,is a zoonotic infectious disease,widespread in all countries in the world.Not only cause infection in poultry,reduced feed efficiency;but also infect humans,against a variety of organs.Therefore,the investigation of fungal diseases in chicken farms,rapid and accurate identification of A.fumigatus,and the establishment of specific,sensitive and rapid diagnostic methods of great significance to the chicken industry and human public health.Chicken sera（287 servings）were collected throughout the year and were tested for（1,3）-β-D-glucan（G test）and galactomannan（GM test）by ELISA.The results of G test showed that the seroprevalence showed a seasonal change with the highest in summer（28.6%）,followed by autumn（12.8%）,winter（12.7%）and spring（12.0%）.GM test results are similar.The results showed that the fungal disease of chicken showed seasonal changes,while summer was the main epidemic season.Isolation of 7 strains of Aspergillus fumigatus from 20 chicken farms in Hebei province.Observe the morphology and extract the DNA of the isolates.PCR amplification was carried out by using the ITS1 and ITS4 common primers of rDNA-ITS gene.The sequences were sequenced and compared with the GenBank database.The results showed that isolates villous,radial growth,colonies central dark green,an white outside.Under the microscope,mycelium was cotton-like,spores pedicel scattered spherical conidia,into a green or dark green.The homology between the isolate and A.fumigatus in GenBank was more than 99.8%,and the homology with the same genus was between 69.4%-85.4%.The seven isolates identified as A.fumigatus by morphological observation combined with rDNA-ITS gene sequence analysis.A.fumigatus was typed using the cell surface protein CSP gene.The results were divided into four genotypes:t04A type（1 strain）,t09^c type（3 strains）,t18^b type（1 strain）and t22^b type（2 strains）.Instilled 1.9×10⁹ CFU/m L isolates’（t09^c type）suspension 100μL to throat of5-day-old chicks.2 pathologic specimen were collected and used for Acid-fast culture and manufacture of biopsies,and HE,PAS,GMS,CFW staining were proceed.The result showed that 3 d after vaccination,chickens had breathing difficulties and other symptoms.Thicken airbag was found after dissected,lung was congested with yellow spots;an isolates of A.fumigatus was found from pathologic specimen;Microscopically,the congestion of the lungs,organizational structure was disappear,lesion has a significant number of hyphae by special stain.These tips to establish a model of A.fumigatus infection in chickens successfully.The isolate（type t09^c）was used to instil 100μL of 1.9×10⁹ CFU/mL bacterial suspension in 5-day-old chickens.Two materials were collected and used as bacteria culture,preparation of pathological sections.The sections were stained with HE,PAS,GMS and CFW.Results The chickens developed dyspnea and other symptoms 3 days after inoculation.The air sacs were thickened and yellow spots were found on the anatomy of the chickens.A.fumigatus was isolated and identified from the disease materials.Under the microscope,pulmonary congestion,tissue structure disappears,there are a large number of mycelium within the special staining lesions.These suggest successfully established A.fumigatus infection in chicken models.Based on the published benAfum gene sequences of Genbank,a pair of specific primers was designed.On this basis,SYBR Green I fluorescence quantitative PCR method was established,and the A.fumigatus isolates were detected by this method.The results showed that the method had high sensitivity and the detection limit of A.fumigatus was 3.76×10¹ CFU/m L;The reproducibility was good with a coefficient of variation of 0.7%-0.8%,no cross-reaction with A.flavus,A.terreus,A.niger,A.tubingensis and C.albicans and other pathogens;The method is simple,time-consuming,the entire test process only 2h.Suggesting that the method can be applied to the rapid detection of A.fumigatus infection.Aiming at the conserved sequences of C.albicans rDNA gene and A.fumigatus benAfum gene,two pairs of specific primers were designed and double fluorescence quantitative PCR（SYBR Green I）was established to detect A.fumigatus and C.albicans.The results showed that there were specific peaks of C.albicans and A.fumigatus at the melting temperatures of 84.5℃-85.0℃and 82.0℃-82.5℃,respectively,and there was no cross-reaction with other fungi.The coefficient of variation of repeated tests between C.albicans and A.fumigatus Respectively 0%,0.2%;the lowest detection concentrations were 4.34×10¹ CFU/mL,3.76×10¹ CFU/mL.This prompted the establishment of double SYBR GreenⅠquantitative PCR method,specific,stable and sensitive,can be used for the detection of A.fumigatus and C.albicans.The reagents for detecting double quantitative PCR of C.albicans and A.fumigatus were assembled into a kit,and the specificity,sensitivity and repeatability of the isolates and diseased samples were tested.The results were good.