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Review 2: "Establishing Methods to Monitor H5N1 Influenza Virus in Dairy Cattle Milk"

While recommending minor clarifications and exploring cost implications, the reviewers emphasize the significance of this research in shaping policies for routine H5N1 monitoring in dairy herds and advancing non-clinical diagnostic approaches for infectious diseases.

Published onJan 30, 2025
Review 2: "Establishing Methods to Monitor H5N1 Influenza Virus in Dairy Cattle Milk"
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Establishing methods to monitor H5N1 influenza virus in dairy cattle milk
Establishing methods to monitor H5N1 influenza virus in dairy cattle milk
Description

Abstract Highly Pathogenic Avian Influenza strain H5N1 has caused a multi-state outbreak among US dairy cattle, spreading across 15 states and infecting hundreds of herds since its onset. We rapidly developed and optimized PCR-based detection assays and sequencing protocols to support H5N1 molecular surveillance. Using 214 retail milk from 20 states for methods development, we found that H5N1 concentrations by digital PCR strongly correlated with qPCR cycle threshold (Ct) values, with dPCR exhibiting greater sensitivity. We also found that metagenomic sequencing after hybrid selection was best for higher concentration samples while amplicon sequencing performs best for lower concentrations. By establishing these methods, we were able to support the creation of a statewide surveillance program to test bulk milk samples monthly from all cattle dairy farms within Massachusetts, which remain negative to date. The methods, workflow, and recommendations described here provide a framework for others aiming to conduct H5N1 surveillance efforts.

RR\ID Evidence Scale rating by reviewer:

  • Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.

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Review: This is a comprehensive tour de force of lab work using a large collection of samples. I liked the exhaustive comparison of qPCR and dPCR, and extraction kits (and links to other testing modalities like using wastewater testing kits), optimisation of primer/probe concentrations, and sequencing approaches. Although the statewide testing was negative, there is ample data on the sensitivity of their method, as well as a solid discussion section, to be reassured this was indeed real. Most optimisation was done with spiked-in synthetic RNAs (not real virus) but samples containing natural virus were utilised.

Use of more standard influenza assays such as those targeting M segment, as well as standard whole genome amplification protocols using one set of primers, would have made this relevant to a wider community (but may have reduced sensitivity). The WGS methods would have been interesting to compare alongside the tiled amplicon methods, especially with the noted downsides of them in biases of certain amplicons.

There are a few points where additional details/explanation would have helped clarify minor points:

  • Can the PCR assay distinguish high versus low pathogenicity (i.e presence of polybasic cleavage site) or is it solely recent clade H5?

  • Graph formats were inconsistent and distracting at times

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