Detection of A(H5N1) influenza virus nucleic acid in retail pasteurized milk

doi:10.21203/rs.3.rs-4572362/v1

The detection of A(H5N1) clade 2.3.4.4b influenza A virus (IAV) in dairy cattle in the U.S. presents a significant threat to the economy, animal well-being, and public health. Preliminary reports of a high viral load in milk from infected cattle sparked concern regarding the safety of the commercial milk supply in the U.S.1–3 Through a convenience sampling of pasteurized milk purchased from retail stores across the United States, we detected A(H5N1) viral nucleic acid in 36.3% of samples. Viral sequencing confirmed the presence of genetic material from A(H5N1) clade 2.3.4.4b influenza A viruses. Despite the detection of relatively high levels of viral RNA, no evidence of viable virus was found following inoculation of MDCK cells, embryonated chicken eggs, or mice with positive retail milk samples. This study highlights the wider distribution of states affected by the virus than suggested by official reports, provides supporting evidence for the efficacy of pasteurization, and suggests the value of retail milk as a means to monitor continued viral evolution.

Biological sciences/Microbiology/Virology/Influenza virus

Biological sciences/Microbiology/Virology/Viral epidemiology

Since the January 2022 detection of A(H5N1) clade 2.3.4.4b influenza A viruses (IAV) in avian species in the U.S, the viruses have infected over 90 million domestic birds (commercial and backyard poultry) across 48 U.S states posing a significant threat to the economy, animal well-being, and public health.⁴All IAV are segmented viruses with an affinity for mutation and reassortment, which contribute to their continuous transmission, evolution, and genetic diversity. Wild birds are reservoirs for IAV, playing a substantial role in the dissemination of the virus through migration.^5–8 A(H5N1) clade 2.3.4.4b viruses were detected in the Netherlands in October 2020, emerging from a reassortment event with A(H5N8) and Eurasian wild bird IAV.⁹ The A(H5N1) 2.3.4.4b viruses have exhibited extensive geographical spread across multiple continents, infecting and contributing to considerable mortality events in a diverse range of species including wild birds, poultry, and terrestrial and marine mammals.^10–14

The recent geographical and evolutionary expansion of highly pathogenic avian influenza (HPAI) A(H5Nx) viruses, coupled with frequent spillover from wild birds to mammals, has sparked concerns about potential mammalian adaptation. Since 1997, 890 human cases of H5 influenza have been reported with a 50% case-fatality rate, highlighting a notable absence in protective immunity in the global human population.¹⁵ While avian-origin viruses typically lack key adaptations for human infection and onward transmission, experimental studies have revealed a subset of A(H5Nx) viruses capable of binding to both human α2,6-linked and avian α2,3-linked sialic acid host receptors.^16,17 Further investigation into the role of mammalian adaptation is crucial, as it holds implications for human infection and potential human-to-human transmission.

In 2024, A(H5N1) clade 2.3.4.4b virus was identified in U.S. dairy cattle in Texas and has now been confirmed in 10 U.S. states including Texas, Kansas, New Mexico, South Dakota, North Carolina, Ohio, Michigan, Colorado, and Iowa.³ The rapid spread to multiple U.S. states shortly after detection suggests that cattle-to-cattle transmission is occurring, likely facilitated by cattle movements.¹⁸ Infected cattle exhibited a notable decline in milk production; simultaneously mortalities among wild birds and domestic cats in the affected areas were also noted, prompting immediate concern.¹ The severity of clinical signs in infected cattle has been variable, from mild to lethal (personal communication). More consistently reported, however, was the presence of high viral loads in milk.^1–3 To date, only three confirmed human cases have been identified in connection with the current outbreak in dairy cattle: all in farm workers, two of whom presented with conjunctivitis.^4,19 The introduction of the virus into dairy cattle creates additional pathways for exposure at the human-animal interface, increasing the risk to public health. Currently, there is no fully implemented surveillance program for farm workers at the frontline of exposure, which may lead to additional human cases going undetected. However, safety precautions and personal protective equipment for farm workers and those in direct contact with A(H5N1) infected dairy cattle has been made available.^20,21

The lack of an established surveillance program for farm workers leaves many questions unanswered regarding the risk of infection due to exposure to milk from A(H5N1) clade 2.3.4.4b infected dairy cattle. Concerns have also been raised regarding the safety of the commercial milk supply and the efficacy of pasteurization for virus inactivation. Therefore, to understand the extent of human exposure to the bovine A(H5N1) virus in both occupationally exposed people, i.e., farm workers and the general public, milk surveillance is necessary. Here, we present our findings on the detection of A(H5N1) viral RNA in retail pasteurized milk, representing milk processing plants in 18 U.S. states.

Detection of HPAI A(H5N1) clade 2.3.4.4b in pasteurized milk

Influenza A virus (IAV) was detected in 36.3% (61/168) of pasteurized milk samples purchased from retail stores across the U.S. by rRT-PCR and confirmed by SJCRH as subtype A(H5N1). Among positive milk samples at initial IAV rRT-PCR screening, Ct values ranged from 23.7- 36.9 and the average copy number per qPCR was 635.5 copies/µL. Total sample representation encompasses milk processing plants across 18 U.S. states. However, A(H5N1) positive milk samples were identified from 20 different processing plants across 10/18 states, including 5 states where no outbreaks in dairy cattle have been reported. The location of dairy cattle farms providing milk to processing plants is unknown.

Genetic analysis of HPAI A(H5N1) clade 2.3.4.4b in pasteurized milk

We selected the 24 milk samples with the lowest Ct values and attempted viral sequencing. Viral sequences were generated from all 24 samples with all generated consensus sequences, mostly representing partial genomes, having highest nucleotide similarity to bovine A(H5N1) viruses. While we refer to these sequences here as consensus, it should be noted that we do not know if the sequences were all derived from the same virus or represent a mixture of closely related viruses present in the milk sample. We were able to generate full length HA sequences from 12 samples. These HA sequences from pasteurized retail milk clustered phylogenetically within the clade of publicly available bovine virus sequences and the human A/Michigan/90/2024 (H5N1) virus confirming their identify as 2.3.4.4b viruses (Figure 1). The full genomes were generated from four samples. The concatenated genomes of the consensus genomes from these samples again clustered phylogenetically with the bovine and related 2.3.4.4b viruses and belonged to genotype B.3.13 (Figure 2).

Virus Isolation

No cytopathic effect (CPE) was observed from cells inoculated with any milk sample or negative control medium at any time point, and no HA activity was detected at 72 hpi. In contrast, our positive control, A/bovine/TX inoculated cells exhibited marked CPE ≥ 48 hpi, and supernatants had HA activity of 64 HA units/50 µL. Allantoic fluid from 72 hpi milk-inoculated eggs was also negative for HA activity 72 hpi. Pooled samples (n = 2 or 3 HA negative samples per poll) were subjected to a second round of passage in both cells and eggs for 72 hpi and were also negative for CPE and HA activity, respectively.

Inoculation of Mice with Milk Samples

All mice intranasally inoculated with rRT-PCR positive (Ct 23.7 to 28.0) milk showed no signs of clinical disease or mortality. A transient decrease in weight (≤ 7% starting weight) was observed in 3 mice (0077, 0085, 0060) 1 dpi after milk inoculation, but quickly resolved 24 hours later and all mice continued to gain weight until the end of the study. In contrast, A/bovine/OH virus-inoculated mice (positive controls) exhibited a gradual decrease in body weight and observable clinical illness as early as 1 dpi; all animals succumbed to infection 4 – 6 dpi (Figure 3).

To understand if sufficient antigen was present in milk samples to generate an immune response, surviving animals’ sera were subjected to HAI assay 14 dpi. No animals seroconverted to the A/bovine/Texas antigen. To confirm the absence of an immune response, animals were re-challenged with a lethal dose of A/bovine/Ohio. All animals exhibited rapid weight loss beginning 3 dpi and succumbed to infection 4 – 5 dpi.

Early reports of A(H5N1) in cow milk have been documented^1–3,18, raising concerns about the safety of the U.S. commercial milk supply. In the present study, we detected A(H5N1) viral nucleic acid in 36.3% of pasteurized milk samples purchased from retail stores across the U.S. Despite the detection of relatively high levels of viral RNA, no evidence of viable virus was found following inoculation of MDCK cells, embryonated chicken eggs, or mice with retail positive milk samples. Taken together, our findings indicate that the outbreak is more widespread than currently reported, but pasteurization is working as an effective tool to inactivate virus in the milk.

Retail milk is a composite, commingled sample sourced not only from numerous individual cattle within a singular farm, but hundreds to thousands of cattle across multiple dairy farms. Consensus genome sequences derived from retail milk represent a population-level pooled sample, requiring careful interpretation and more stringent consideration for inclusion in phylogenetic analyses. We acknowledge the value of retail milk as a genomic surveillance tool to monitor viral evolution and propose the following nomenclature for strain names: A/bovine/location/MILK-process-XXX/2024. If a single state for “location” is unclear or unknown, USA is appropriate, while “process” may be replaced with the 2-digit state postal code corresponding to the milk processing plant. The sequence information we generated from the 24 samples with highest viral load confirmed that the RNA detected was derived from viruses genetically similar to those 2.3.4.4b viruses infecting cows.

Currently, our understanding of pathogenesis and evolution is limited following the detection of A(H5N1) clade 2.3.4.4b in dairy cattle. While there is documented evidence of systemic, respiratory, and neurologic disease in terrestrial mammals^11,13, this study provides a precedent in exploring tissue tropism within the mammary tract, underscoring the risk of milk as a vehicle for the spread of the virus. The high viral load observed in pasteurized milk samples, as indicated by the low Ct values in this study and relatively high copy numbers, implies that we may be underestimating the number of cattle infected. Furthermore, it suggests that the distribution of the virus is likely more extensive than currently reported based on multiple surrounding states with positive milk plants (Figure 4). While retail milk may originate from numerous independent dairy cattle producers, which may not directly correspond with the state of the milk processing plant, this discovery carries implications for the magnitude of the outbreak.

Extensive research studying zoonotic transmission of IAVs highlight that swine are mixing vessels, sharing avian-receptors (SAα2,3Gal) and mammalian-receptors (SAα2,6Gal) for the virus.²² Additionally, swine have an established human-animal interface through agricultural activities. Of major concern is the spread of A(H5N1) cattle-to-swine, increasing the potential for zoonoses. Notably, mutation analyses conducted on cattle and feline samples positive for A(H5N1), revealed amino acid sites associated with mammalian adaptation and a greater affinity for human-type receptors^2,18. These results combined with reports of co-expression of human and avian receptors in the mammary glands of infected dairy cattle²³, highlight the potential role of cattle as mixing vessels for zoonotic IAVs. Questions remain regarding infected dairy cattle as a source of IAV infection for susceptible, unprotected hosts who are directly or indirectly exposed to milk or secretions.

None of the retail milk samples included in this study contained viable, infectious A(H5N1) virus per in vitro and in vivo assays, which provides evidence that pasteurization is sufficient for inactivation of pathogens in the commercial milk supply. However, should infectious A(H5N1) be found in the commercial milk supply, the finding would have serious implications on the economy and human health. As the scope of the U.S. outbreak continues to expand with more herds becoming infected and increased spillover events identified, active surveillance programs are critical in cattle, wild birds, and humans at the frontline of exposure. Through active surveillance and data sharing, we can more accurately assess the magnitude of the outbreak, evolution of the virus, and potential for human-to-human transmission.

Sample collection

Between April 13-May 3, 2024, 168 unique pasteurized milk samples were purchased from retail stores in 12 U.S. states: California (CA), Colorado (CO), Florida (FL), Illinois (IL), Kansas (KS), Michigan (MI), Minnesota (MN), Missouri (MO), New Mexico (NM), Ohio (OH), Oklahoma (OK), and Texas (TX). In the field, each sample was aliquoted immediately into a 15 mL conical and a 50 mL conical, frozen on dry ice, and shipped to The Ohio State University (OSU) where they were stored at –80°C until used for further diagnostic testing. For data analysis purposes, we attempted to include milk samples that were unique by milk processing plant and/or expiration date.

PCR Screening

All samples received at OSU were screened for the presence of influenza A viral nucleic acid using real-time reverse transcription polymerase chain reaction (rRT-PCR). Using the original 15 mL aliquot for each sample, viral RNA was extracted from a 200 μl volume using the MagMAX viral/pathogen II nucleic acid isolation kit (Life Technologies, cat#A48383). An rRT-PCR, targeting the IAV matrix gene, was performed using the VetMAX-Gold SIV detection kit (Life Technologies, Cat# 4415200) with Xeno internal positive control to validate the extraction process and PCR reaction. Any sample with a cycle threshold (Ct) value of ≤ 40 was considered positive and the original 50 mL aliquot, preserved at -80°C, was shipped to St. Jude Children’s Research Hospital (SJCRH) for confirmation of subtype. To assess viral load, all positive samples were also tested at OSU via quantitative polymerase chain reaction (qPCR) with the VetMAX-Gold SIV detection kit as described previously. The standard curves were constructed by 4-fold serial dilutions of the SIV-Xeno RNA control mix in a concentration from 1.0e⁴ to 1.5e^-1 (copies/μl) in duplicate. The reactions were run on the ABI 7500 FAST Real-Time PCR system for 45 cycles.

Subtyping

To subtype the IAV detected in the samples, 50 mL aliquots from the PCR screened IAV positive retail milk samples were received on dry ice at SJCRH and stored at 4°C until processing. RNA from each sample was extracted from a 200uL volume using Qiagen RNeasy Mini Kit (Cat# 74106). From each extraction, 2uL of RNA per reaction was run using ABI TaqMan Fast Virus 1-Step Master Mix (Cat# 4444436) in a 20uL reaction on an ABI 7500 FAST Real-Time PCR system for 40 cycles. Each sample was run in duplicate (FluA-Matrix²⁴) or triplicate (Influenza H5b²⁵) with primers and probe sequences from designs by US CDC.

Minion Library preparation and viral sequencing from milk

RNA was extracted from milk samples using TRIzol LS Reagent (Life technologies) per manufacturer’s protocol. Using Invitrogen Superscript VI One-Step RT-PCR with Platinum Taq (Thermo Fisher Scientific), extracted RNA was amplified with a pool of Uni12, MBTuni-12, MBTuni-13, and pairs of influenza segment-specific primers. Equimolar amounts of purified amplicons were subjected to DNA repair using NEBNext Ultra II End repair/dA-tailing Module (New England BioLabs, MA), followed by individual ligation with native Barcoding Kit 96 V14 (SQK-NBD114.96) using NEBNext Quick Ligation Module (New England BioLabs, MA). Barcoded samples were pooled and subjected to further purification using Agencourt AMPure XP Beads (Beckman Coulter) before adaptor ligation by NEBNext Quick Ligation Module (NEB). Prepared libraries were adjusted to 20 femtomole before loading into the spot-on port on the MinION Mk 1 B flow cells (Oxford Nanopore Technologies).

Genetic analysis

ABLASTN analysis was conducted for assembled HA sequences from this study and closely related sequences downloaded from the Global Initiative on Sharing All Influenza Data (GISAID; downloaded June 6, 2024). The assembled sequences underwent multiple sequence alignment and trimming using BioEdit 7.2. MEGA 11 was used for construction of HA phylogenetic trees by applying the Neighbor-Joining method and Kimura 2-parameter model with 1000 bootstrap replicates.

To build a concatenated phylogenetic tree generated from all eight viral RNA segments, all publicly available A(H5N1) viruses from North America with collection dates from January 2024 through May 2024 (n=350) were downloaded from GISAID. Four full genome consensus sequences that we generated from milk samples were included in the data set. The eight segments were concatenated and underwent multiple sequence alignment by MAFFT and trimming using BioEdit 7.2 to remove noncoding regions. A phylogenetic tree was constructed using maximum-likelihood methods available in IQ-TREE 2.3.4 with a GTR + G model of nucleotide substitution. The constructed concatenated tree was visualized in FigTree v.1.4.4. Genotype classification was determined using the GenoFLU tool.

Virus isolation from A(H5N1) rRT-PCR positive milk samples

To determine virus viability in pasteurized milk samples obtained from retail stores, virus isolation was attempted on 61 samples using tissue culture and egg propagation methods. Madin-Darby Canine Kidney cells (MDCKs, ATCC, P35) were plated at 5 x 10⁵ cells/well in 12-well plates in growth medium (MEM, 5% fetal bovine sera, 1x penicillin/streptomycin/amphotericin B, 1mM L-glutamine) overnight, resulting in confluent monolayers. MDCKs (n = 2 wells/milk sample) were washed 3x with sterile phosphate buffered saline (PBS) and inoculated with 1 mL milk sample prepared neat (undiluted) for 1 hr at 37 °C in infection medium (MEM, 1% bovine serum albumin, 1x penicillin/streptomycin/amphotericin B, 1mM L-glutamine). Negative control wells included 1 mL infection medium only, and positive control wells were inoculated with A/bovine/Texas/98638/2024 (H5N1, A/bovine/TX) (≈ MOI 0.01). Inocula were removed, the monolayers were washed 3x with PBS, and the supernatants were replaced with 1mL infection medium. Cells were incubated for 72 hr, 37°C. Every 24 hr, monolayers were observed for influenza-induced cytopathic effect (CPE). Supernatants were collected at 72 hours post-inoculation (hpi) and assessed for hemagglutination activity (HA assay) as an indicator of virus particle presence, using chicken red blood cells (cRBCs).

Virus isolation was also attempted in 10-day old embryonated chicken eggs. Eggs (n = 3/milk sample) were inoculated with 200 µL milk diluted 1:1 in egg injection antibiotics (2 x 10⁵ U/mL penicillin G potassium salt, 40,000 U/mL streptomycin, 20,000 U/mL Polymixin B, 4 mg/mL gentamicin in PBS). Eggs were candled daily for 72 hr to assess viability. At 72 hpi, allantoic fluid was harvested from each

Both cell supernatants and egg allantoic fluid from the initial round of isolation (Passage 1, P1) were subjected to a second-round blind passage. Supernatants or allantoic fluid that were HA-negative after P1 were pooled (n = 3 samples/pool) and inoculated to MDCKs or injected into eggs as described above. Both cell and egg viability were monitored every 24 hr and HA activity was assessed at 72 hpi.

Inoculation of mice with retail milk samples

Mice at six weeks of age (BALB/c (Jackson Labs, median weight 17.6g)) were inoculated with 6 retail milk samples (n = 3 mice/sample) representing diversity in region of sale, milk distributor, and low Ct value (23.7 to 28.0) (Figure 3, Table 1). Mice were lightly anesthetized with 4% isoflurane and inoculated with 30 µL undiluted milk intranasally (IN). Positive control mice were inoculated with 1 x 10⁶ TCID₅₀ units of A/bovine/Ohio/ B24OSU-439/2024 (H5N1, A/bovine/OH). Weight and clinical disease (disheveled coats, lethargy, anorexia, and/or neurological involvement characterized by tremors/dystonia/limb paralysis) were assessed daily. At 14 days post-inoculation (dpi), sera was collected from the mice and tested for antibodies via hemagglutination inhibition assay (HAI) as described previously²⁶. Mice were re-challenged with 2 x 10⁵ TCID₅₀ A/bovine/OH and monitored for weight loss and mortality for an additional 5 days.

Data visualization was generated using FigTree v.1.4.4, Prism v.10.2.3, and ArcMap (ESRI). Descriptive statistics were calculated using SAS Studio v.3.81.

Ethics Statement/Facilities

Experiments using mice were approved by the St. Jude Children’s Research Hospital Institutional Animal Care and Use Committee (IACUC, protocol number 428) in accordance with the guidelines established by the Institute of Laboratory Animal Resources, approved by the Governing Board of the US National Research Council. In vivo and virus isolation experiments were conducted by trained personnel in a United States Department of Agriculture (USDA)-inspected Animal Biosafety Level 3+ animal facility following regulations outlined by the Division of Agricultural Select Agents and Toxins (DASAT) at the USDA Animal and Plant Health Inspection Service (APHIS), as governed by the United States Federal Select Agent Program (FSAP) regulations (7 CFR Part 331, 9 CFR Part 121.3, 42 CFR Part 73.3).

Data Availability

The figure for the distribution of states affected by the A(H5N1) outbreak in dairy cattle was generated using data available from the United States Department of Agriculture. (https://www.aphis.usda.gov/livestock-poultry-disease/avian/avian-influenza/hpai-detections/livestock). All publicly available A(H5N1) viruses from North America with collection dates from January 2024 through May 2024 (n=350) were downloaded from GISAID for inclusion in phylogenetic analyses. All other data are included in this article and its supplementary files.

Code Availability

Not applicable

Acknowledgements

Through collaboration in the Centers of Excellence for Influenza Research and Response (CEIRR) Risk Assessment working groups, we would like to thank Martha Nelson and Brian Wasik for their contributions to the proposed nomenclature.

This work was supported by the Centers of Excellence for Influenza Research and Response, National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH), Department of Health and Human Services, under contract 75N93021C00016 (St. Jude Center of Excellence for Influenza Research and Response).

Author Contributions

Conceptualization: JL, RJW, ASB

Methodology: NNT, JCJ, JF, AK, RJW, ASB

Investigation: all

Visualization: NNT, JCJ, AK

Funding Acquisition: RJW, ASB

Project Administration: JL, RJW, ASB

Writing, original draft: NNT, JCJ, JF, RJW, ASB

Writing, editing/review: all

*Correspondence to Andrew Bowman ([email protected])

Competing Interests

The authors declare no competing interests.

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Table 1. 61 rRT-PCR positive retail milk samples used in inoculation of MDCK cells, embryonated chicken eggs, or mice.

Retail Milk Strain Name	Date of Sample Collection	Milk Processing Plant State	IAV Screen Ct	Copy Number (copies/ μl)	Sequence Obtained?
A/bovine/USA/MILK-48-24MK0001 /2024	4/13/2024	Texas	33.4	10.3	Not obtained
A/bovine/USA/MILK-48-24MK0002 /2024	4/13/2024	Texas	30.5	65.4	Not obtained
A/bovine/USA/MILK-48-24MK0009 /2024	4/13/2024	Texas	29.7	147.3	Not obtained
A/bovine/USA/MILK-48-24MK0012 /2024	4/13/2024	Texas	30.4	86.4	Not obtained
A/bovine/USA/MILK-08-24MK0015 /2024	4/13/2024	Colorado	29.8	221.6	Not obtained
A/bovine/USA/MILK-48-24MK0016 /2024	4/14/2024	Texas	35	7.7	Not obtained
A/bovine/USA/MILK-48-24MK0021 /2024	4/14/2024	Texas	31.1	77.7	Not obtained
A/bovine/USA/MILK-08-24MK0022 /2024	4/14/2024	Colorado	28.2	531.4	Not obtained
A/bovine/USA/MILK-48-24MK0024 /2024	4/14/2024	Texas	30.5	46.8	Not obtained
A/bovine/USA/MILK-48-24MK0027 /2024	4/14/2024	Texas	31.7	85.3	Not obtained
A/bovine/USA/MILK-48-24MK0028 /2024	4/14/2024	Texas	29.4	101.7	Not obtained
A/bovine/USA/MILK-48-24MK0030 /2024	4/14/2024	Texas	29.8	126.3	Not obtained
A/bovine/USA/MILK-48-24MK0032 /2024	4/14/2024	Texas	26.2	727.3	Full genome
A/bovine/USA/MILK-48-24MK0033 /2024	4/14/2024	Texas	36.5	0.9	Not obtained
A/bovine/USA/MILK-48-24MK0034 /2024	4/14/2024	Texas	32.6	40.7	Not obtained
A/bovine/USA/MILK-48-24MK0035 /2024	4/14/2024	Texas	26.2	668.5	Full HA
A/bovine/USA/MILK-48-24MK0036 /2024	4/14/2024	Texas	26.2	796.5	Full genome
A/bovine/USA/MILK-48-24MK0039 /2024	4/14/2024	Texas	30.6	41.7	Not obtained
A/bovine/USA/MILK-48-24MK0040 /2024	4/14/2024	Texas	29.3	86.2	Not obtained
A/bovine/USA/MILK-48-24MK0043 /2024	4/14/2024	Texas	33.5	20.4	Not obtained
A/bovine/USA/MILK-48-24MK0044 /2024	4/14/2024	Texas	27.6	608.4	Not obtained
A/bovine/USA/MILK-48-24MK0045 /2024	4/14/2024	Texas	28.0	473.5	Not obtained
A/bovine/USA/MILK-48-24MK0050 /2024	4/14/2024	Texas	32.9	66.9	Not obtained
A/bovine/USA/MILK-48-24MK0051 /2024	4/14/2024	Texas	35.9	2.1	Not obtained
A/bovine/USA/MILK-18-24MK0055 /2024	4/14/2024	Indiana	36.9	0.2	Not obtained
A/bovine/USA/MILK-48-24MK0057 /2024	4/15/2024	Texas	26.5	436.7	Full HA
A/bovine/USA/MILK-05-24MK0060 /2024	4/15/2024	Arkansas	23.7	3771.0	Full HA
A/bovine/USA/MILK-0-24MK0061 /2024	4/15/2024	Arkansas	29.7	329.0	Not obtained
A/bovine/USA/MILK-48-24MK0062 /2024	4/15/2024	Texas	28.1	257.2	Not obtained
A/bovine/USA/MILK-40-24MK0068 /2024	4/15/2024	Oklahoma	25.7	1364.0	Partial sequence
A/bovine/USA/MILK-08-24MK0070 /2024	4/15/2024	Colorado	28.6	292.8	Not obtained
A/bovine/USA/MILK-20-24MK0071 /2024	4/15/2024	Kansas	27.1	975.0	Partial sequence
A/bovine/USA/MILK-18-24MK0074 /2024	4/15/2024	Indiana	33.2	41.3	Not obtained
A/bovine/USA/MILK-48-24MK0077 /2024	4/15/2024	Texas	24.8	1450.0	Full genome
A/bovine/USA/MILK-20-24MK0081 /2024	4/15/2024	Kansas	36.4	0.8	Not obtained
A/bovine/USA/MILK-20-24MK0082 /2024	4/15/2024	Kansas	28.4	282.9	Not obtained
A/bovine/USA/MILK-20-24MK0083 /2024	4/15/2024	Kansas	25.0	1941.3	Full HA
A/bovine/USA/MILK-20-24MK0084 /2024	4/15/2024	Kansas	26.0	797.6	Full HA
A/bovine/USA/MILK-20-24MK0085 /2024	4/15/2024	Kansas	24.8	2950.7	Full HA
A/bovine/USA/MILK-40-24MK0089 /2024	4/15/2024	Oklahoma	25.2	1610.2	Full HA
A/bovine/USA/MILK-40-24MK0090 /2024	4/15/2024	Oklahoma	25.7	2585.0	Partial sequence
A/bovine/USA/MILK-20-24MK0092 /2024	4/15/2024	Kansas	32.3	42.3	Not obtained
A/bovine/USA/MILK-20-24MK0093 /2024	4/15/2024	Kansas	27.2	1681.4	Not obtained
A/bovine/USA/MILK-26-24MK0104 /2024	4/15/2024	Kansas	25.0	7236.1	Full genome
A/bovine/USA/MILK-26-24MK0107 /2024	4/15/2024	Michigan	35.7	2.3	Not obtained
A/bovine/USA/MILK-08-24MK0109 /2024	4/15/2024	Colorado	29.3	389.2	Not obtained
A/bovine/USA/MILK-20-24MK0111 /2024	4/15/2024	Kansas	28.1	744.7	Not obtained
A/bovine/USA/MILK-20-24MK0112 /2024	4/15/2024	Kansas	27.8	1747.6	Not obtained
A/bovine/USA/MILK-39-24MK0115 /2024	4/16/2024	Ohio	37.0	6.3	Not obtained
A/bovine/USA/MILK-48-24MK0126 /2024	4/16/2024	Texas	35.6	3.9	Not obtained
A/bovine/USA/MILK-20-24MK0129/2024	4/16/24	Kansas	26.3	3526.4	Partial sequence
A/bovine/USA/MILK-27-24MK0135 /2024	4/18/2024	Minnesota	33.9	24.7	Not obtained
A/bovine/USA/MILK-29-24MK0146 /2024	4/13/2024	Missouri	27.6	1220.5	Partial sequence
A/bovine/USA/MILK-39-24MK0147 /2024	4/13/2024	Ohio	28.3	1045.8	Partial sequence
A/bovine/USA/MILK-08-24MK0150 /2024	4/13/2024	Colorado	28.9	1171.6	Partial sequence
A/bovine/USA/MILK-26-24MK0166 /2024	4/20/2024	Michigan	32.7	70.6	Not obtained
A/bovine/USA/MILK-26-24MK0167/2024	4/20/2024	Michigan	31.6	316.8	Not obtained
A/bovine/USA/MILK-26-IL-M09 /2024	4/16/2024	Michigan	23.6	Not determined	Not obtained
A/bovine/USA/MILK-26-IL-M10 /2024	4/16/2024	Michigan	24.7	Not determined	Not obtained
A/bovine/USA/MILK-48-24MK0176 /2024	5/3/2024	Texas	35.0	101.0	Not obtained
A/bovine/USA/MILK-AR001/2024	4/20/2024	Arkansas	28.0	Not determined	Full HA

Table 2. Positive detections of H5N1 in milk samples reported by state where milk was processed using rRT-PCR results.

Milk Processing Plant State	# H5 positive samples	# Total samples (%)	% Positive
Arkansas	3	3 (1.79)	100.0
Arizona	0	5 (2.98)	-
CALIFORNIA	0	3 (1.79)	-
COLORADO	5	20 (11.90)	25.0
GEORGIA	0	1 (0.60)	-
INDIANA	2	21 (12.50)	9.5
KANSAS	12	15 (8.93)	80.0
Kentucky	0	1 (0.60)	-
MARYLAND	0	1 (0.60)	-
MICHIGAN	5	16 (9.52)	31.25
MINNESOTA	1	7 (4.17)	14.3
MISSOURI	1	5 (2.98)	20.0
NEBRASKA	0	4 (2.38)	-
NEW YORK	0	10 (5.95)	-
OHIO	2	18 (10.71)	11.1
OKLAHOMA	3	3 (1.79)	100.0
TEXAS	27	33 (19.64)	81.8
VIRGINIA	0	2 (1.19)	-
Total	61	168	36.3

There is NO Competing Interest.

SupplementaryTable1.xlsx
Dataset 1

Detection of A(H5N1) influenza virus nucleic acid in retail pasteurized milk

Status:

Version 1

Abstract

Figures

Introduction

Results

Discussion

Materials And Methods

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

Version 1