الفهرس 
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Abstract
The present study was undertaken to investigate the prevalence of fungal infection in internal organs of broiler chicken and their environment and evaluation the antifungal potentials of some oils and zinc nanoparticles on the growth of fungal isolates in addition to monitoring the gene sequencing of fungal isolates and comparing gene expression before and after applying oils and zinc oxide nanoparticles treatments on the fungal isolates.
The mycological examination of a total 227 broiler chickens samples (20 crop, 80 air sac, 80 pericardium, 20 liver and 27 proventriculus), revealed that the incidence of fungi in samples was (21%). A total of 49 fungal isolates were recovered from samples included 20 yeast isolates (8.8%) which identified as C. albicans (8 isolates), C. krusei (3 isolates), C. tropicalis (2isolate) and C. glabrata (2 isolates). While 29 isolates (12.8%) were belong to mycelial fungi which were A. niger (10 isolates), A. flavus (6 isolate), A .fumigatus (5 isolates), Penicillium (4 isolates) and A. nidulans, A. terrus, Cladosporum and Zygomycetes (1 isolate for each species). Regarding fungal contamination of individual internal organs of chickens, from 20 samples of crops and 80 air sac samples, 7 fungal isolates (35%) and 17 fungal isolates (21.25%) were recovered respectively. While, from (80 pericardium, 20 liver and 27 proventriculus samples), 10 isolates (12.5%), 5 fungal isolates (25%) and 10 fungal isolates (37%) were isolated respectively.
A total 29 isolates of mold (12.8%) were recovered from 227 samples of internal organs of chickens. The isolated fungi were identified as A. niger (10 isolates)(4.4%), A. flavus (6 isolate) (2.6%), A. fumigatus( 5 isolates)(2.2%)and Penicillium (4 isolates)(1.8%) . While, A. nidulans, A. terrus, Cladosporum and Zygomycetes were recovered from (0.4%) of samples (1 isolate for each species)
A total of 22 fungal isolates were recovered from 47 samples from broilers feed, water and bedding. The total incidence of fungi in samples was 46.8% included 12 isolates (25.5%) namely A. flavus 4 isolates (33.3%), A. fumigatus 3 isolates (6.4), Zygomycetes 2 (2.3%), A. niger, A. nidulans and A. terrus (1 isolate for each)(2.1% ) were yielded from samples. Ten isolates of yeasts (21.3%) were recovered from all environmental samples and identified as C. albicans (5 isolates; 50%), C.krusei (2 isolates; 20%) and C. tropicalis (3 isolates; 30%).
A total of 25 yeast isolates (15 recovered from chickens and 10 from chicken environment) were identified biochemically. The isolates were characterized as Candida species including 13 isolates C. albicans, 5 C. krusei, 5 C. tropicalis and 3 C. glabrata.
The evaluation of antifungal potential of carvacrol oil at a concentration of (0.01%, 0.1%, 0.25%, 0.5%. and 1%) against C. albicans, A. niger, A. flavus, A.nidulans, A. fumigatus and Penicillium were investigated. The results showed that the minimal inhibitory concentration of carvacrol was (0.1%) which completely inhibited the growth of all examined isolates.
The antifungal potential of cinnamic acid at gradual concentration of (0.01%, 0.1%, 0.25%, 0.5%. and 1%) against C. albicans, A. niger, A. flavus, A. nidulans, A. fumigatus and Penicillium were investigate. The results showed that the minimal inhibitory concentration of cinnamic acid was (0.1%) which completely inhibited the growth of all examined isolates.
The chemical synthesis of ZnO-NPs were proceeded and were characterized for their optical and nano structural properties by Scanning Electron Microscope and FT-IR spectra (Fourier Transform Infrared Spectrometer which showed that the particles morphology was spherical and granular in shape of ZnO- NPs and that they are well crystallized in the nano-size of (50-60 nanometer scale) as observed by Transmission electron micrograph (SEM). Whereas the UV-Vis absorption spectrum of ZnO sample in the range 200-550 nm. It showed excitonic peaks with absorbance intensity very close to wave length of 360 nm.
In the present work, the prepared ZnO NPs was evaluated for its antifungal activities against the isolated fungi from broiler chicken and their environment using agar dilution method. The results illustrated that MIC of ZnO NPs for more pathogenic fungi for chickens (A. flavus and C. albicans) were (300 and 200 ug/ml), respectively.
As the used concentration of ZnNPS increased (0, 50, 100, 200, 300 and 400ug/ml), the colony count of treated fungi were decreased till complete inhibition of fungal growth.
When the treated fungi were subjected to SEM, the damage and rupture of their cell wall were detected in the area of surrounding growth media. The normal conidial cell of yeast or mould has a spherical shape and smooth cell wall and intact cell membrane of conidia. The effect of inhibitory concentration of ZnO nanoparticles on the treated fungi was observed as membrane damage of cells and some pits that have been caused in inter cellular components, leading to leakage and finally cell death.
In the present work, the treated A. flavus and C. albicans by zinc oxide nanoparticles and oils (carvacrol, cinnamic acid) were evaluated by molecular detection for the changes in gens density and structure before and after treatments. All treated isolates of A. flavus and C. albicans were inhibited their ability for growth as detected by agar diffusion method. However, the extraction of DNA from these treated isolates showed some differences, where the corresponding genes detected by PCR method in spite the same isolates were showed no growth and completely inhibited their viability in diffusion tests. While, in other cases the genes of treated strains were eliminated after treatment. These results indicated the efficacy of control by zinc oxide nanoparticles and oils carvacrol and cinnamic acid which caused some changes or in-activation in gene. The detection of A. flavus DNA by PCR reaction before and after treatments with oils or zinc nanoparticles showed that no any bands of genes in case of treatment with oils carvacrol and cinnamic acid. Whereas the treatment with ZnNPs showed sever reduction in density of PCR band to 10% in comparison with the non-treated A. flavus band.
from the foregoing results it could be concluded that the fungal pathogens were reported as potential organisms and caused different diseases conditions in human poultry and animals, particularly after prolonged exposure to adverse environmental condition. Therefore, the essential significance of this study is the indication that natural oils as carvacrol and cinnamic acid and ZnO nanoparticles to inhibit the growth of mold and yeast which cause superficial or deep infections or even toxicities in chickens. In addition, they could be used in the field of human and veterinary medicine as a fungicide in successful treatment of microbial diseases. In addition, the synthesis preparation of Nano-Zn described here is cost and effective, environmentally friendly and non infectious for industrial workers. The antimicrobial effects of ZN-Nanoparticles are due to the damage of the cell wall of the microbial cells leading to leakage of the cell contents and finally cell death. Advanced field application of the present findings in direct treatment of diseased animal and poultry as well as synergistic therapy of NPs with other traditional herbal oils and antibiotics drugs was urgently required to decrease the used concentration of nanoparticles, overcome the microbial resistant to traditional antibiotics and resulted more efficient antimicrobial activity for the treatment of human and animal diseases.