#qRTPCR #Detection of Inactivated #H5 Avian #Influenza Virus in Raw #Milk Samples by Miniaturized Instruments Designed for On-Site Testing

 

Abstract

Highly pathogenic avian influenza virus (HPAIV) of H5 and H7 subtypes has emerged as one of the most important zoonotic pathogens in the 21st century with significant economic consequences. The recent outbreak of H5N1 avian influenza (AI) in dairy cattle highlighted the importance of early detection in managing and mitigating HPAIV outbreaks. A successful high-speed diagnostic response requires rapid site and specimen access, minimal time for test protocols, and prompt communication of the diagnostic results to government officials. A new diagnostic paradigm that consists of miniaturized extractor and qPCR instruments (EZextractor and EZcycler MiniQ), designed for mobile, on-site testing has been compared with a platform of benchtop instruments (QIAGEN RNeasy and QuantStudio 5) for detecting inactivated H5 avian influenza virus (AIV) spiked in raw milk samples. Two sets of experiments were performed: 1) 15 raw milk samples, obtained from 15 different farms, diluted with phosphate-buffered saline and spiked with the virus to reach approximately 10 copies/mcL virus concentration, and 2) raw milk samples from two farms, each spiked with the inactivated AIV H5 followed by 5 series of dilution to reach AIV concentrations of 1000, 100, 10, 1 and 0.1 copies/mcL. Results show that despite the inhibitors in raw milk, AIV in all samples can be detected by both platforms. The MT platform showed higher sensitivity than the benchtop platform: the Ct values from the MT were ~2 units lower than the benchtop Ct values. Our findings demonstrate the robustness of the MT platform for diagnosing AIV H5 in raw milk samples and support its use as an on-site diagnostic for rapid surveillance and response.

Competing Interest Statement

The authors have declared no competing interest.

Funder Information Declared

DiaVac Biotech Co.

Schweitzer Biotech Co.

Source: 

Link: https://www.biorxiv.org/content/10.1101/2025.06.02.657307v3

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Retail #Milk #Monitoring of #Influenza #H5N1 in Dairy #Cattle, #USA, 2024–2025

 

Abstract

US retail milk monitoring during April 13–May 3, 2024, identified influenza A(H5N1) viral RNA in 36% of retail milk samples, indicating widespread undetected infections in US dairy cows. After federal initiatives, reported infections more closely aligned with findings in retail milk during December 27, 2024–January 29, 2025, reflecting improved detection and control.

Source: 

Link: https://wwwnc.cdc.gov/eid/article/32/2/25-1332_article

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Inactivation of avian #influenza virus in #yogurt made from raw #milk

 

Highlights

• Fermentation of raw milk for 7 h at 42 °C to pH 4.4 reduced AIV levelsAIV by ≤ 4.1 log10 EID50.

• Incubation of raw milk for 7 h at 42 °C to pH 6.6 reduced levels by ≤ 4.3 log10 EID50.

• PH, temperature and/or bacterial degradation appreciably reduced AIV levels in raw milk.

Abstract

In March 2024, highly pathogenic avian influenza (HPAIV) H5N1 was first detected in U.S. dairy cattle and has since spread to herds across at least 17 states. Infected cows typically present with mastitis, decreased milk production, and poor milk quality with high viral loads in milk. While commercial pasteurization of milk effectively inactivates avian influenza virus (AIV), growing consumer interest in raw milk and derived products raises public health concerns due to the risk of zoonotic transmission. Standard yogurt production includes an initial heating step at 82 °C for 30 min to denature milk proteins which also inactivates AIV. However, some home yogurt recipes omit this initial heating step. This project determined whether AIV present in raw milk could remain viable through fermentation and persist in the final yogurt product. Raw milk (ca. pH 6.7) was spiked with AIV (ca. 6.6 log10 50 % egg infectious doses (EID50) per mL and inoculated with a commercial starter culture to produce yogurt. The viability of the virus was determined before and after fermentation (ca. 7.3 h) at 42 °C with resultant pH drop ≤4.4. A significant (p < 0.05) reduction of viable AIV (≥4.1 log10 EID50) was observed in both the yogurt and the control samples of raw milk incubated at 42 °C but without starter culture (ca. pH 6.63). Viral inactivation was likely due to a combination of incubation at a sublethal temperature, pH below 4.4, and microbial degradation. Thus, properly fermented yogurt has a negligible risk of transmitting AIV to humans.

Source: 

Link: https://www.sciencedirect.com/science/article/pii/S0740002025002576?via%3Dihub

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Quantitative #risk #assessment of #human #H5N1 #infection from consumption of fluid cow's #milk

 

Abstract

The spillover of H5N1 clade 2.3.4.4b into dairy cattle has raised concerns over the safety of fluid milk. While no foodborne infection has been reported in humans, this strain has infected at least 70 people and milk from infected cows is known to be infectious by ingestion in multiple other species. Investigation into the public health threat of this outbreak is warranted. This farm-to-table quantitative microbial risk assessment (QMRA) uses stochastic models to assess the risk of human infection from consumption of raw and pasteurized fluid cow's milk from the United States supply chains. These models were parameterized with literature emerging from this outbreak, then employed to estimate the H5N1 infection risk and evaluate multiple potential interventions aimed at reducing this risk. The median (5th, 95th percentiles) probabilities of infection per 240-mL serving of pasteurized, farmstore-purchased raw, or retail-purchased raw milk were 7.66E-19 (2.39E-20, 4.02E-17), 1.56E-7 (6.67E-10, 1.28E-5), and 1.40E-7 (6.65E-10, 1.13E-05), respectively. Our results confirm that pasteurization is highly effective at reducing H5N1 infection risk. Scenario analysis revealed quantitative real-time reverse transcriptase-polymerase chain reaction (qrRT-PCR) testing of bulk tank milk to be an effective method for numerically reducing risk from raw milk. Additionally, we identify knowledge gaps related to human H5N1 dose-response by ingestion and raw milk consumption patterns. These findings emphasize the importance of mechanistic epidemiologic models for informing public health responses amidst outbreaks with foodborne potential and highlight the need for additional research into raw milk consumption patterns to better understand this exposure pathway.

Competing Interest Statement

The authors have declared no competing interest.

Funding Statement

Research reported in this publication was supported by the Office of the Director, National Institutes of Health of the National Institutions of Health (NIH) under Award Number T32ODO011000 to KK. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Research Resources or the NIH. Additionally, this research was partially supported by grants to RI from the National Institute of Food and Agriculture, USDA, Hatch under Accession Number 7000433, as well as Multistate Research Funds Accession Number 1016738.

Source: 

Link: https://www.medrxiv.org/content/10.1101/2024.12.20.24319470v3

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#Stability of Avian #Influenza #H5N1 Virus in #Milk from Infected #Cows and Virus-Spiked Milk

 

{Excerpt}

To the Editor:

In March 2024, public health agencies in the United States reported highly pathogenic avian influenza (HPAI) virus of subtype A(H5N1) in dairy cattle, with virus detected in milk samples from symptomatic animals. Viral genetic fragments were subsequently found in pasteurized retail dairy products, which aroused concern about potential exposure through the human food supply. Heat treatment has been shown to reduce or eliminate infectious virus from both milk from infected cows and milk spiked with HPAI A(H5N1) virus. However, whether HPAI A(H5N1) virus in spiked milk from healthy cows replicates the properties of virus in milk from infected cows is unclear. We directly compared the stability of HPAI A(H5N1) virus in infected cows’ milk with that in spiked milk at 4°C (refrigeration temperature) and after heat treatment at 63°C (low-temperature, longer-duration pasteurization) or 72°C (high-temperature, shorter-duration pasteurization). (...)

(...)

Source: 

Link: https://www.nejm.org/doi/10.1056/NEJMc2502494

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#Influenza A Virus detection in Bulk Tank and Pen Level #Milk from #Dairies Affected by Highly Pathogenic Avian Influenza #H5N1

 

Abstract

Highly pathogenic avian influenza virus H5N1 has been infecting dairy herds in the U.S. since its initial incursion into cows in early 2024. Although national strategies have aimed to detect affected herds, the best way to surveil herds for the H5N1 virus has not been formally studied and we also do not understand herd-level patterns of infection. To understand infection patterns of H5N1 in dairy herds over time, we conducted early surveillance of non-affected farms in California in the Fall of 2024 in an observational study. Daily bulk tank milk (BTM) samples were submitted from each herd and tested for influenza A (IAV) via rRt-PCR. In a subset of herds, IAV testing of multiple excretion types from cattle of different classes and pen-level daily milk was also completed soon after BTM detection. Daily detections of IAV occurred in BTM for a minimum of 33 days, with some herds continuing to have detection beyond a 75-day window. BTM Ct nadirs were seen between 1-3 weeks of detection. In herds that were tested, virus was detected in the milk from all pens of cattle within a very short time frame after BTM detection, or prior to the initiation of pen level sampling. A very low percentage (2.8%) of individual cow samples tested positive for IAV when collected soon after BTM detection, and although the virus was found in all excretion types, a majority of positive samples were from milk. This suggests that BTM may be the best early indicator of herd infection, and that movement of the virus to all lactating pens of cattle after herd incursion is relatively quick. These results also suggest that surveillance strategies with a long interval between BTM testing days may miss herds with short infection windows. Because most herds experienced test days where some submitted BTM samples had virus detected while others did not, and virus was detected in pen level milk samples when the BTM from the herd had become test negative, this work also highlights the necessity of studying the test sensitivity of IAV rRt-PCR detection in aggregate milk samples.

Competing Interest Statement

The authors have declared no competing interest.

Funding Statement

This project was completed using Federal funds provided through the United States Department of Agriculture, Cooperative Agreement 25-9419-0731 and the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No. 75N93021C00016.

Source: MedRxIV, https://www.medrxiv.org/content/10.1101/2025.10.26.25338833v1

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Highly Pathogenic Avian #Influenza #H5N1 in Raw #Pet #Foods and #Milk: A Growing #Threat to both Companion Animals and #Human #Health, and Potential Raw Pet Food Industry Liability

 

Highlights

• Raw pet foods and raw milk are emerging sources of H5N1 in pets.

• Cats are more severely infected with H5N1 when compared to dogs.

• H5N1 persistence in mammals indicate adaptive variants with increased zoonotic potential.

• No reported pet-to-human transmission of H5N1 has been reported.

• FDA now requires RMBD makers who are covered under FSMA to assess HPAI risk.

Abstract

The increasing popularity of raw meat-based diets (RMBDs) and raw milk feeding in companion animals presents a growing concern for zoonotic disease transmission. Recent evidence has demonstrated that these products can serve as vehicles for highly pathogenic avian influenza (HPAI) H5N1, an emergent viral threat with a host range from birds, dairy cattle, and pets to humans. Since the emergence of clade 2.3.4.4b in 2020, HPAI H5N1 has caused widespread outbreaks in poultry, wild birds, and mammals, including dairy cattle and cats. Transmission to pets has been linked to ingestion of contaminated raw pet food and unpasteurized milk. Notably, multiple outbreaks in cats across Europe, Asia, and North America have been associated with raw pet food products, while recent U.S. cases confirm direct viral transmission from infected pet food, raw milk, and colostrum. Experimental studies have also supported the plausibility of gastrointestinal and respiratory routes of infection in cats and dogs, with felines appearing particularly susceptible, often exhibiting severe clinical disease and high mortality. A number of documented recalls of H5N1-contaminated raw pet food and raw milk in the US underscore the persistence of infectious viruses in cold-stored food products and highlight the risks of feeding raw diets. Although pet-to-human transmission of the HPAI H5N1 virus has not been reported yet, cat-to-human transmission of the H7N2 influenza virus has been reported in the USA. This review presents current evidence on H5N1 in RMBDs and raw milk, its epidemiology in companion animals, outbreaks, and the health implications among pets and humans. By raising awareness among pet owners, industry stakeholders, and veterinarians, this paper highlights the immediate need for stringent surveillance and improved biosecurity in raw food supply chains to minimize viral transmission risks thereby safeguarding pet health and curb the potential spillover to humans.

Source: Journal of Food Protection, https://www.sciencedirect.com/science/article/pii/S0362028X25001802?via%3Dihub

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Pasteurized #Milk Serves as a Passive #Surveillance #Tool for Highly Pathogenic Avian #Influenza Virus in Dairy #Cattle

 

Abstract

The emergence of H5N1 highly pathogenic avian influenza virus (HPAIV) clade 2.3.4.4b in dairy cattle across multiple U.S. states in early 2024 marks a major shift in the virus’s host range and epidemiological profile. Traditionally limited to bird species, the ongoing detection of H5N1 in cattle, a mammalian host not previously considered vulnerable, raises urgent animal and human health concerns about zoonoses and mammalian adaptation. We assessed the feasibility of using commercially available pasteurized milk as a sentinel matrix for the molecular detection and genetic characterization of H5N1 HPAIV. Our aim was to determine whether retail milk could serve as a practical tool for virological monitoring and to evaluate the use of full-length genome segment amplification for extracting genomic sequence information from this highly processed matrix. Our results link HPAIV sequences in store-bought milk to the cattle outbreak and highlight both the potential and the limitations of retail milk as a surveillance window. Together, these findings provide evidence that influenza A virus RNA can be repeatedly detected in retail milk in patterns linked to specific supply chains, with genomic data confirming close relationships with the viruses circulating in cattle.

Source: Viruses, https://www.mdpi.com/1999-4915/17/10/1318

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Repeated #oral #exposure to #H5N1 #influenza virus in pasteurized #milk does not cause adverse responses to subsequent influenza #infection

 

Abstract

In March 2024, a highly pathogenic avian influenza H5N1 (HPAI) clade 2.3.4.4b virus was identified in US dairy cows, with spillover to cats, poultry, and humans. Up to 30% of commercial pasteurized milk tested contained viral genome copies. The impact of residual viral remnants on host immunity is unknown. Orally ingested proteins can stimulate gut-associated lymphoid tissues, potentially inducing tolerance and altering responses to later infection. We found that milk pasteurization fully inactivated pandemic H1N1 and bovine H5N1 influenza viruses yet preserved hemagglutinin (HA) protein integrity. In mice, repeated oral exposure to inactivated virus did not alter mortality after H5N1 virus challenge. Preliminary data showed that naïve mice exposed to improperly pasteurized milk containing live H5N1 virus developed lethal infection, whereas prior H1N1 infection conferred protection. Mice with preexisting H1N1 immunity remained protected when challenged with bovine H5N1 virus after exposure to H5N1 pasteurized in milk. These findings suggest that pasteurized milk containing inactivated H5N1 virus poses minimal health risks.

Source: Science Advances, https://www.science.org/doi/10.1126/sciadv.aeb3906

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The (#digestive) path less traveled: #influenza A virus and the #gastrointestinal tract

 

ABSTRACT

Influenza A virus (IAV) infection of the respiratory tract can cause both respiratory and non-respiratory symptoms. Gastrointestinal (GI) symptoms such as diarrhea, vomiting, and abdominal pain can occur in persons with seasonal influenza A or novel IAV infections, but the extent to which IAVs can infect and replicate in GI tissues is understudied. The ongoing outbreak of A(H5N1) IAV in US dairy cattle associated with sporadic human infections has highlighted the potential public health threat posed by the introduction of infectious virus into materials that may be consumed by humans, such as milk. Here, we review epidemiologic reports documenting the frequency of GI complications in humans infected with seasonal and novel IAVs and present laboratory studies supporting the capacity of IAV to replicate in mammalian GI tissues, with an emphasis on A(H5N1) viruses. Studies assessing the ability of IAV to cause mammalian infection following consumption of virus-containing material are also presented. Collectively, these studies suggest that gastric exposure represents a potential non-respiratory route for A(H5N1) IAVs in mammals that can lead to infection and support that IAV may be detected in mammalian intestinal tissues following multiple exposure routes.

Source: mBio, https://journals.asm.org/doi/full/10.1128/mbio.01017-25?af=R

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#Influenza #infection of the mammary #gland

 

ABSTRACT

The mammary gland is an essential organ for milk production, providing essential immune and nutritional support to offspring and supplying dairy products for human consumption. In both humans and animals, the lactating mammary gland is susceptible to bacterial and viral infections, which can lead to mastitis and, in some cases, vertical transmission to offspring, with potential adverse effects on infant health. However, until recently, the role of respiratory viruses in mammary gland infection has been relatively understudied, particularly their ability to infect mammary epithelial cells and transmit through lactation. The recent emergence of highly pathogenic avian influenza H5N1 clade 2.3.4.4b in dairy cattle has demonstrated the virus’s capacity to replicate in the mammary gland, cause mastitis, and produce high viral loads in milk. This raises significant concerns about the potential for zoonotic transmission to humans and other animals in contact with infected dairy cows and unpasteurized milk. In this mini-review, we highlight key studies that demonstrate the replication of influenza and other viruses in the mammary gland, summarize recent findings from experimental and natural H5N1 clade 2.3.4.4b infections in dairy cows and small animal models, and discuss the broader One Health implications of the current H5N1 outbreak. We emphasize the urgent need for an interdisciplinary collaboration across sectors to mitigate the risks posed by influenza viruses with pandemic potential.

Source: Journal of Virology, https://journals.asm.org/doi/full/10.1128/jvi.01940-24?af=R

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#Inactivation of #Influenza A Viruses (#H1N1, #H5N1) During #Grana-Type Raw #Milk #Cheesemaking: Implications for Foodborne #Transmission Risk

Abstract

Background: 

The detection of H5N1 highly pathogenic avian influenza virus (HPAIV) in lactating dairy cattle in the United States, with high viral titers in raw milk, has raised concerns about potential zoonotic transmission through the consumption of unpasteurized milk and raw-milk dairy products. While inactivation studies exist for pasteurized milk, data on virus persistence during the manufacture of raw-milk cheeses remain scarce. 

Aim: 

To evaluate the survival and inactivation of avian influenza viruses (AIV), including both low pathogenic (LPAIV, H1N1) and highly pathogenic (HPAIV, H5N1) strains, during the production and ripening of Grana-type hard cheeses made from raw bovine milk. 

Methods: 

Experimental cheesemaking was conducted using raw milk artificially contaminated with A-Duck-Italy-281904-2-06 (H1N1; 10 7,75 EID50 per mL) or A-Duck-Italy-326224-2-22 (H5N1 clade 2.3.4.4b; 10 6,75 EID50 per mL). Cheeses were produced in accordance with Parmigiano Reggiano production standards and ripened for 30 days at 5 or 6 degrees Celsius. Viral presence was assessed in finished cheeses by inoculation on SPF embryonated chicken eggs (ECE), hemagglutination (HA) assay, and monoclonal antibody-based ELISA. 

Results: 

No infectious virus was detected in any cheese sample produced from contaminated milk following two blind passages in SPF-ECE. Both HA and ELISA tests yielded negative results, indicating complete inactivation of the virus. 

Conclusion: 

This study demonstrates that the traditional Grana-type cheese production process, including curd cooking, acidification, and ripening, effectively inactivates both LPAIV and HPAIV, even at high contamination levels. These findings support the microbiological safety of hard cheeses made from raw milk with regard to AIV, contributing to risk assessment and food safety policies during avian influenza outbreaks.

Source: BioRxIV, https://www.biorxiv.org/content/10.1101/2025.06.18.660327v1

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qRTPCR #Detection of Inactivated #H5 Avian #Influenza Virus in Raw #Milk Samples by Miniaturized Instruments Designed for On-Site Testing

Abstract

Highly pathogenic avian influenza virus (HPAIV) of H5 and H7 subtypes has emerged as one of the most important zoonotic pathogens in the 21st century with significant economic consequences. The recent outbreak of H5N1 avian influenza (AI) in dairy cattle highlighted the importance of early detection in managing and mitigating HPAIV outbreaks. A successful high-speed diagnostic response requires rapid site and specimen access, minimal time for test protocols, and prompt communication of the diagnostic results to government officials. A new diagnostic paradigm that consists of miniaturized extractor and qPCR instruments (EZextractor and EZcycler MiniQ), designed for mobile, on-site testing has been compared with a platform of benchtop instruments (QIAGEN RNeasy and QuantStudio 5) for detecting inactivated H5 avian influenza virus (AIV) spiked in raw milk samples. Two sets of experiments were performed: 1) 15 raw milk samples, obtained from 15 different farms, diluted with phosphate-buffered saline and spiked with the virus to reach approximately 10 copies/mcL virus concentration, and 2) raw milk samples from two farms, each spiked with the inactivated AIV H5 followed by 5 series of dilution to reach AIV concentrations of 1000, 100, 10, 1 and 0.1 copies/mcL. Results show that despite the inhibitors in raw milk, AIV in all samples can be detected by both platforms. The MT platform showed higher sensitivity than the benchtop platform: the Ct values from the MT were ~2 units lower than the benchtop Ct values. Our findings demonstrate the robustness of the MT platform for diagnosing AIV H5 in raw milk samples and support its use as an on-site diagnostic for rapid surveillance and response.

Source: BioRxIV, https://www.biorxiv.org/content/10.1101/2025.06.02.657307v2

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Amplicon #sequencing of pasteurized retail #dairy enables genomic #surveillance of #H5N1 avian #influenza virus in #USA #cattle

Abstract

Highly pathogenic avian influenza (HPAI) viruses with H5 hemagglutinin (HA) genes (clade 2.3.4.4b) are causing an ongoing panzootic in wild birds. Circulation of these viruses is associated with spillover infections in multiple species of mammals, including a large, unprecedented outbreak in American dairy cattle. Before widespread on-farm testing, there was an unmet need for genomic surveillance. Infected cattle can shed high amounts of HPAI H5N1 viruses in milk, allowing detection in pasteurized retail dairy samples. Over a 2-month sampling period in one Midwestern city, we obtained dairy products processed in 20 different states. Here we demonstrate that a tiled-amplicon sequencing approach produced over 90% genome coverage at greater than 20x depth from 5 of 13 viral RNA positive samples, with higher viral copies corresponding to better sequencing success. The sequences clustered phylogenetically within the rest of the cattle outbreak sequences reported. A combination of RT-qPCR testing and sequencing from retail dairy products can be a useful component of a One Health framework for responding to the avian influenza outbreak in cattle.

Source: PLoS One, https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0325203

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qRT-PCR #Detection of Inactivated #H5 Avian #Influenza Virus in Raw #Milk Samples by Miniaturized Instruments Designed for On-Site Testing

Abstract

Highly pathogenic avian influenza virus (HPAIV) of H5 and H7 subtypes has emerged as one of the most important zoonotic pathogens in the 21st century with significant economic consequences. The recent outbreak of H5N1 avian influenza (AI) in dairy cattle highlighted the importance of early detection in managing and mitigating HPAIV outbreaks. A successful high-speed diagnostic response requires rapid site and specimen access, minimal time for test protocols, and prompt communication of the diagnostic results to government officials. A new diagnostic paradigm that consists of miniaturized extractor and qPCR instruments (EZextractor and EZcycler MiniQ), designed for mobile, on-site testing has been compared with a platform of benchtop instruments (QIAGEN RNeasy and QuantStudio 5) for detecting inactivated H5 avian influenza virus (AIV) spiked in raw milk samples. Two sets of experiments were performed: 1) 15 raw milk samples, obtained from 15 different farms, diluted with phosphate-buffered saline and spiked with the virus to reach approximately 10 copies/mcL virus concentration, and 2) raw milk samples from two farms, each spiked with the inactivated AIV H5 followed by 5 series of dilution to reach AIV concentrations of 1000, 100, 10, 1 and 0.1 copies/mcL. Results show that despite the inhibitors in raw milk, AIV in all samples can be detected by both platforms. The MT platform showed higher sensitivity than the benchtop platform: the Ct values from the MT were ~2 units lower than the benchtop Ct values. Our findings demonstrate the robustness of the MT platform for diagnosing AIV H5 in raw milk samples and support its use as an on-site diagnostic for rapid surveillance and response.

Source: BioRxIV, https://www.biorxiv.org/content/10.1101/2025.06.02.657307v1

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Stability of #influenza viruses in the #milk of #cows and #sheep

Abstract

In late 2023, H5N1 high pathogenicity avian influenza (HPAIV) started circulating in dairy cattle in the USA. High viral titres were detected in milk from infected cows, raising concerns about onwards human infections. Although pasteurisation was shown to effectively inactivate influenza viruses in milk, unpasteurised milk still poses a risk of infection, both from occupational exposure in dairies and from the consumption of raw milk. We therefore assessed how long influenza viruses could remain infectious for in milk without heat inactivation. We examined the stability of a panel of influenza viruses in milk, including a contemporary H5N1 HPAIV and a variety of other influenza A and D viruses. We incubated viruses in cows' milk under laboratory conditions: at room temperature to simulate exposure in dairies and at 4°C to simulate exposure to refrigerated raw milk. Following an isolated report of H5N1 viral RNA being detected in milk from a sheep in the UK, we also carried out similar experiments with a laboratory strain of IAV in sheep's milk. Although the survival of influenza viruses in milk was variable, we consistently found that under laboratory conditions substantial viral infectivity remained over periods when people might reasonably be exposed to infected milk - for over a day at room temperature and for more than 7 days when refrigerated. Our results highlight the zoonotic risk of H5N1 HPAIV in raw milk from infected animals and reinforce the importance of taking measures to mitigate this risk.

Source: MedRxIV, https://www.medrxiv.org/content/10.1101/2025.05.28.25328508v1

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Establishing #Methods to #Monitor #Influenza A #H5N1 Virus in Dairy #Cattle #Milk, #Massachusetts, #USA

Abstract

Highly pathogenic avian influenza A(H5N1) virus has caused a multistate outbreak among US dairy cattle, spreading across 16 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 samples from 20 states for methods development, we found that H5N1 virus concentrations by digital PCR strongly correlated with quantitative PCR cycle threshold values; digital PCR exhibited greater sensitivity. Metagenomic sequencing after hybrid selection was best for higher concentration samples, whereas amplicon sequencing performed best for lower concentrations. By establishing these methods, we were able to support the creation of a statewide surveillance program to perform monthly testing of bulk milk samples from all dairy cattle farms in Massachusetts, USA, which remain negative to date. The methods, workflow, and recommendations described provide a framework for others aiming to conduct H5N1 surveillance efforts.

Source: US Centers for Disease Control and Prevention, https://wwwnc.cdc.gov/eid/article/31/13/25-0087_article

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Impact of #pH and #temperature in dairy #processing on the infectivity of #H5N1 avian #influenza viruses

Abstract

Highly pathogenic avian influenza viruses (HPAIV) of subtype H5N1 (clade 2.3.4.4b) have crossed the species barrier and caused a mastitis-like infection in dairy cows. The high levels of infectious virus found in the milk raised considerable concerns about the safety of raw milk products. This study examined the effect of temperature and pH on the stability of HPAIV and low-pathogenic avian influenza viruses (LPAIV). We found that H5N1 HPAIV remained infectious in milk at 4 degrees Celsius for four weeks, with slow decreases at 21 degrees Celsius, and complete inactivation at 37 degrees Celsius after four weeks. H5N1 LPAIV was stable at 50 degrees Celsius for 30 minutes but inactivated at higher temperatures (55 degrees Celsius for 10 minutes, 60 degrees Celsius for 1 minute, or 72 degrees Celsius for 30 seconds). At pH levels between 6 and 10, the virus remained stable but was partially inactivated at pH 5.0 and completely inactivated at pH 4.0. During yogurt production, H5N1 LPAIV was completely inactivated when the pH reached 4.3. In cheese production, the lowest pH reached was between 5.0 and 5.3. When H5N1 LPAIV was incubated with soft and semi-hard cheese for one day at 4 degrees Celsius, infectious virus titers decreased by 5.1 and 3.9 log10, respectively. When H5N1 LPAIV was incubated with buffer adjusted to pH 5.0, infectious virus titer dropped by only 3.3 log10, suggesting that, alongside pH, other processes of cheese ripening likely influence virus stability. In conclusion, H5N1 avian influenza viruses are largely inactivated during lactic acid fermentation of raw milk. Future studies will assess the required cheese ripening time for complete inactivation.

Source: BioRxIV, https://www.biorxiv.org/content/10.1101/2025.03.21.644501v1

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Highly Pathogenic Avian #Influenza A(#H5N1) Virus #Stability in Irradiated Raw #Milk and #Wastewater and on #Surfaces, #USA

Abstract

We measured stability of infectious influenza A(H5N1) virus in irradiated raw milk and wastewater and on surfaces. We found a relatively slow decay in milk, indicating that contaminated milk and fomites pose transmission risks. Although the risk is low, our results call for caution in milk handling and disposal from infected cattle.

Source: US Centers for Disease Control and Prevention, https://wwwnc.cdc.gov/eid/article/31/4/24-1615_article

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Examining the #Survival of A #H5N1 #Influenza Virus in Thermised Whole #Cow #Milk

Abstract

The recent spillover events of highly pathogenic avian influenza (HPAI) A(H5N1) clade 2.3.4.4b to dairy cattle, and high viral shedding in the milk from infected animals, has created concern that milk and dairy products could be a route for human infection. It has been demonstrated that pasteurization is effective in inactivating A(H5N1) in milk. However, multiple dairy products are made with unpasteurized but thermised milk. The aim of this study was to examine whether some conditions commonly used for thermisation are effective against inactivation of A(H5N1) in whole milk. For this purpose, we artificially inoculated whole raw cow milk with 6.5 log10 EID50 A(H5N1) and heated for 15 seconds at 60°C, 63°C and 66°C, the viral infectivity was tested using embryonated chicken eggs. We observed over 4 and 5 log10 reduction in viral infectivity at 60°C and 63°C, respectively. The viral infectivity was reduced to below the detection limit at 66°C. We also calculated the D-values, the time required to reduce the viral titer by one log10, for each treatment and as expected, we observed a decrease in D-values with increasing thermisation temperature. These data demonstrate that thermisation is effective in reducing the viral load and thus they allow for informed risk assessment of A(H5N1) contaminated dairy products made from thermized milk.

Source: BioRxIV, https://www.biorxiv.org/content/10.1101/2025.03.05.641644v1

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