Additional sheep were coinfected with equal numbers of progenitor and passaged strains (1000 CFUs in total) following the procedure described above (table S4). We introduced a single restriction site synonymous mutation in the noncoding region of a pseudogene in the progenitor strains: the vWbp pseudogene in NCTC8325 and the arlR pseudogene in N315. These mutations were used to track the abundance of progenitor and passaged strains in the milk sampled from the coinfected ewes. From the isolation plates, at least 10 colonies were sampled for polymerase chain reaction (PCR) amplification and restriction enzyme digestion of the relevant genomic region. In addition, two isogenic strains, NCTC8325 and NCTC8325s (synonymous SNP in the vWbp pseudogene), were used to coinfect 20 additional sheep by inoculating 1000 CFUs of each of their mammary glands. For every week during 10 weeks, PCR amplification and restriction digestion with Eco RI were performed on 10 colonies isolated from the milk samples.

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