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The battle against mastitis remains an enduring challenge in contemporary animal farming. This paper presents the frequency of bacterial pathogens isolated from milk samples collected on 3 farms with a previous history of clinical mastitis in the Mačva region during the year 2022. A total of 77 Simmental cows were enrolled in the study, ranging from the first to sixth lactation and managed in a tie-stall system. The results obtained indicate that the frequency of clinical mastitis in the animals considered was 16.9%. Of a total of 13 pathogenic isolates with clinically present mastitis, Staphylococcus aureus was recorded in 5 (38.4%), Streptococcus uberis in 3 (23.1%), Escherichia coli in 2 (15.4%), Klebsiella pneumoniae in 2 (15.4%), and Streptococcus dysagalactiae in 1 (7.7%). Notably, the largest number of clinical mastitis cases (namely 8, or 61.5%) was recorded during the winter period. Both cases of mastitis caused by K. pneumoniae were detected in the summer, the occurrence of which was implicated in changes in the bedding straw. According to the stage of lactation, the highest frequency of mastitis was found in the early stage of lactation totaling 8 cases (61.5%), followed by 4 (30.8%) in the middle phase of lactation, and only 1 (7.7%) in the late stage of lactation. The causative agents of the Streptococcus genus showed susceptibility to β lactamates in all the cases, whereas all of the E. coli and K. pneumoniae isolates were found resistant to ampicillin, amoxicillin with clavulanic acid.

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