One hundred and seventy-seven (177) bulk tank milk samples were analyzed with a commercially available real-time polymerase chain reaction kit and 11 (6.21%), 41 (23.16%), and 58 (32.77%) tested positive for Mycoplasma bovis, Staphylococcus aureus, and Streptococcus agalactiae, respectively. Statistical analysis revealed a significant relationship between the presence of S. aureus and S. agalactiae. Enumeration of somatic cells was performed in the same samples by flow cytometry. The somatic cell counts were found higher in S. aureus and S. agalactiae positive samples. No association was found between M. bovis presence and somatic cells counts. Low internal assay control Ct values were found to be related with high somatic cell counts. Noticeably, this is the first report for the presence of M. bovis in Cyprus. Therefore, its presence was confirmed by bulk tank milk culture, conventional PCR, and next generation sequencing. Furthermore, M. bovis was typed with multilocus sequencing typing and was allocated to sequence type 29 (ST 29). Real-time PCR in bulk tank milk samples is a useful tool to detect mammary infections, especially for neglected pathogens such as M. bovis.

Recent evidence suggests that the lower gut microbiome of ruminants presents roles in their health and environment, including the development of the mucosal immune system, milk production efficiency and quality and subsequent methane emissions. However, there are proportionately fewer studies on this complex microbial community in cattle and region-focus studies are non- existent. Herein, we present the research protocol of the GUTBIOME CY project pertaining to determine the composition of the lower gut microbiome in dairy cows situated in 37 farms across five districts of the island of Cyprus. Detailed questionnaires on animal husbandry and farming practices will be gathered from each farm. Faecal, milk (individual and bulk) and water samples will also be collected from cows and their offspring. Samples will be analysed using a combination of molecular biology and bioinformatics pipelines to define microbiome profiles and antimicrobial resistance (AMR). Information collected from the questionnaires will be use to test for associations between animal husbandry or farming practices and microbiome components and AMR. Collected samples will establish the first dairy cattle biobank in the country for contributing substantially towards scientific advancements in microbiome research and providing insights to all stakeholders, tailored to the unique agricultural context of Cyprus.