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Abstract Three hundred milk samples of different animal species including buffaloes (100), cows (100), goats (50) and ewes (50) were collected from Assiut City farms as well as 50 raw marketable milk samples were purchased from Assiut City shopkeepers and street vendors. The samples were examined for subclinical mastitis and for isolation and identification of Mycoplasma species. In case of buffaloes’ milk samples, the incidence of subclinical mastitis were 55 and 59% based on CMT and MWST, respectively and Mycoplasma could be isolated from 8% of the examined samples. Moreover, the percentage of the contaminated subclinically mastitic samples was 5.5 and 2.2% of non mastitic milk samples were positive for Mycoplasma. Biochemical identification of the isolated strains declared that, M. bovirhinis and / or M. species group 7 were the isolated strains. The results revealed that 61% of the examined cow’s milk samples were subclinically mastitic by using CMT and 68% were positive for subclinical mastitis by using MWST. However, Mycoplasma could not be detected in the evaluated samples. The percentages of subclinical mastitis by using CMT and MWST in the examined cow’s milk samples were 61 and 68%, respectively. In addition, most of subclinically mastitic milk samples were weak (42.6%) followed by distinct, and strong mastitically (18.0 and 1.7%) based on CMT and 72.0, 26.3 and 1.7% by using MWST. However, Mycoplasma could not be detected in the tested samples. Concerning goat’s milk, sucblinical mastitis could be detected in 90% of the investigated samples based on CMT and MWST. In addition, 8% of the examined samples were contaminated with Mycoplasma and all of positive samples were subclinically mastitic. M. mycoides subsp. mycoides LC and M. capricolum were the identified strains biochemically. By using CMT and MWST for detection of subclinical mastitis in ewes’ milk samples, it clear evident that, 68% of the tested samples were positive for subclinical mastitis. The incidence of Mycoplasma was 2% and M. capricolum was the identified strain biochemically. The prevalence of Mycoplasma in the investgated marketable milk samples was 2% and the isolated strain was identified as M. bovirhinis and / or M. species. group 7. Moreover, molecular identification of the isolated strains from buffaloes’ milk and marketable milk samples by using specific primer for M. bovirhinis reveled that, no of the tested stain was M. bovirhinis. It is obvious from the result of antibiotic sensitivity test that, the most effective drugs on the isolated strains were the quienolones and nalidixic acid followed by chloramphenicol contrawise, penicillin, glycosides, tetracycline and macrolides have no effect on the tested strains. In addition, UHT cow’s milk and laboratory sterilized goat’s milk were inoculated with the previously isolated and identified Mycoplasma strains to yield a concentration of 106cfu/ml to study the effect of different pasteurization temperatures on the survival of these isolates. The obtained result pinpointed that, M. mycoides subsp. mycoides LC is the most resistant strain to heat treatment, where, it could be detected after 30 min exposure to 63 and 72oC and failed to be detected after 1 min of heat treatment at 85oC. M. capricolum was more sensitive, where it could not survive 1 min exposure to 72 and 85oC, While, M. species. group 7 remained viable even after 30 min of heat treatment at 63oC and 5 min at 72oC. |