Real time PCR for identification of Viable But Non Culturable fungi isolated from mastitic cattle milk samples depending on propidium mono azide stain

Abstract

A total of 150 milk samples were collected from both acute and chronically infected cattle from both Giza and Minoufia governorates. Incidence of mycotic mastitis was at rate of 72% and 88%for Giza and Minoufia governorates respectively. Different mold and yeast species were isolated. The most isolated mold species from Giza samples were Aspergillus species (64.4%) followed by Geotrichum (15.2%)then Penicillium (10.2%), while in Minoufia governorate, the most isolated mold species were Penicillium at a percentage of (50.6%)followed by Aspergillus at a percentage of (40.2%). About yeast species isolation, C. parapsillosisis was the most isolated yeast species from Giza samples with percentage of 33.4%,while in Minoufia samples, C. tropicalis was the most isolated yeast species with a percentage of 22.7%. All of the isolates are identified depending on macroscopical and microscopical identification. Twelve isolated yeast strains were biochemically identified depending on rapaid yeast plus identification system, all of the tested strains were correctly identified except for C. parapsillosis strains, only 83%of the tested strains were correctly identified. Six negative examined milk samples with culture on Sabaroud dextrose agar media were subjected to Rt-PCR and Propidium mono azide stain, four of them were positive although they were negative on culture as they are considered as samples containing VBNC(Viable but non culturable ) strains.