Optimizing Bacteriophage Screening and Isolation Methods for Microbial Samples Derived from Different Body Sites of Cattle

Bacteriophages are increasingly investigated as tools for studying and manipulating microbial communities in cattle. However, phage isolation remains challenging because of host specificity, microbial ecosystem differences, and the lack of optimized screening approaches. The objectives of this study were to (i) optimize the phage-screening method for microbial samples obtained from different cattle body sites, (ii) isolate lytic phages against key bovine pathogens and commensal bacteria, and (iii) characterize the isolated phages and their bacterial hosts. A total of 1214 samples from different cattle body sites (n = 1194) and environmental sources (n = 20) were screened using 13 phage detection methods, including one high-throughput approach. Eighty-three phages were isolated, primarily from ruminal fluid (59), feces (15), vaginal (7) and nasopharyngeal swabs (1), and fetal ruminal fluid (1). The bacterial hosts inhibited by these phages were from 29 genera, with Bacillus (34), Escherichia/Shigella (8), Shouchella (5), Corynebacterium (4), and Lysinibacillus (4) being the most common. No phages were identified against bovine pathogens including Trueperella pyogenes, Mannheimia haemolytica, Pasteurella multocida, or Moraxella bovis. Method 12 demonstrated the highest efficiency in phage recovery, particularly from ruminal samples. The successful recovery of bacteriophages from gastrointestinal, reproductive, respiratory, and fetal bovine samples demonstrates the utility of the optimized screening methods for isolating phages from diverse cattle-associated microbial ecosystems. Further studies are needed to refine these approaches to improve the recovery of phages targeting bovine pathogens.