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Abstract Mastitis remains one of the most common cosmopolitan diseases in the dairy industry leading to great economic losses. Several bacterial species have been identified as mastitis causing pathogens, but many clinical cases of mastitis remain culture negative even after pre-enrichment and enrichment for bacterial growth. This problem causes a difficulty in diagnosis of mastitis and identification of the causative agents and also the conventional culture methods can’t solve the problem due to the absence of growth of causative agents on conventional media. Therefore, the aim of the present study was to throw a glimmer of light on culture negative milk samples collected from clinical cases of mastitis for identifying the undetected causative agents directly through PCR assay followed by sequencing of 16s rRNA gene. In the present work, a total of 171 milk samples were collected from different localities in Sharkia Governorate. The samples included milk from clinical cases of mastitis (137) and raw milk (34). The milk samples from cows and buffaloes showing clinical mastitis were collected from different private farms raised in an open housing system without any hygienic measures and with no previous history of treatment, while the raw milk samples were collected from different supermarkets. Conventional isolation and identification of some Gram negative and some Gram positive bacteria revealed that the most frequently Gram negative bacterial isolate in raw milk samples and milk samples from clinical cases of mastitis was E. coli with. |