Identification of Bovine Meat and Milk Factor (BMMF) DNA in Raw Milk from single Individual Cows from a German experimental cow herd using BMMF-specific primers for PCR and sequencing.
Bovine Meat and Milk Factors (BMMFs) are plasmid-like DNA molecules isolated from bovine milk and serum that have been proposed to contribute to indirect carcinogenesis in humans. BMMF DNA and and increased expression of the conserved Replication protein have been detected in tumor-adjacent mucosa of 99% of colorectal cancer (CRC) patients. Expression was associated with CD68?CD163? macrophages and already increased in cancer-preceding early and late dysplasia. These findings suggest BMMFs as cancer biomarkers and potential contributors to chronic inflammation fueling indirect carcinogenesis. Although BMMF DNA has been found in a wide spectrum of dietary products including commercial pooled milk, the presence and possible distribution of cancer and non-cancer-related BMMF types in the milk of single cows has not been assessed so far. Therefore, in this study, raw milk samples were collected from 18 individual cows using robotic milking systems. DNA was extracted by phenol-chloroform extraction and enriched for circular DNA using rolling circle amplification. BMMF sequences were amplified by PCR using BMMF-specific primers separated by agarose gel electrophoresis, excised, purified, and cloned into pUC19 vector. Sanger sequencing was performed (Eurofins Genomics) for obtaining circular sequences which were analyzed by BLAST to assess identity and similarity to previously described BMMF isolates. BMMF DNA was detected in 16 of the 18 milk samples analysed. In total, 15 distinct BMMF sequences were isolated, of which 4 showed high sequence similarity to previously published BMMF isolates, while 11 are novel sequences that were submitted here. This dataset provides a characterization of the animal-specific BMMF repertoire present in milk from individual cows.