Review 1: "Establishing Methods to Monitor H5N1 Influenza Virus in Dairy Cattle Milk"

RR\ID Evidence Scale rating by reviewer:

• Strong. The main study claims are very well-justified by the data and analytic methods used. There is little room for doubt that the study produced has very similar results and conclusions as compared with the hypothetical ideal study. The study’s main claims should be considered conclusive and actionable without reservation.

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Review: The authors present a noteworthy study that offers valuable insights into the implementation of routine surveillance measures for H5N1 infections in dairy cow herds. They have modified and validated existing methods of molecular diagnostics through detailed and well-documented in silico and in vitro analyses. The study utilizes ready-for-sale cow milk from retail markets as a surrogate for advanced clinical studies, effectively demonstrating the suitability of the validated methodology. A particularly noteworthy aspect of the study is its application in mandatory monthly surveillance studies in Massachusetts, enabling the evaluation of the methodology under real-field conditions.

The manuscript is meticulously crafted, offering precise and clear explanations and statements. However, the reader might have wished for a reference to the anticipated costs of such a surveillance program (e.g., extrapolated to 100 dairy farms). With these guidelines published, it will hardly be possible to circumvent the urgently needed mandatory H5N1 surveillance routines in US dairy herds.

Minor comments:

• The figures for infected herds and affected states should be updated.

• L93: The copy numbers indicated, are they per reaction or per µL?

• L111, 117: “slightly” better, should be defined or statistically supported, otherwise rephrase.