ETIDIOH - NEW

  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 we...

  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...

  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 l...

  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...

  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-contain...

  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 d...

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 wi...

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 phos...

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...

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 phosph...

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 c...

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 p...

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,...

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 ____

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-value...

Abstract The spillover of highly pathogenic avian influenza H5N1 into dairy cattle has raised concerns over the safety of fluid milk . While no human foodborne infection has been reported , this strain has infected dozens of 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 critical . This study uses quantitative risk assessment (QRA) models to represent the United States raw and pasteurized fluid milk supply chains to estimate the risk of human infection from consumption of fluid cow's milk. 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 5.68E-15 (1.77E-16, 2.98E...

Abstract The spillover of highly pathogenic avian influenza (HPAI) H5N1 virus to dairy cows and shedding of high amounts of infectious virus in milk raised public health concerns. Here, we evaluated the decay and thermal stability spectrum of HPAI H5N1 virus in raw milk. For the decay studies , HPAI H5N1 positive raw milk was incubated at different temperatures and viral titers and the thermal death time D-values were estimated. We then heat treated HPAI H5N1 virus positive milk following different thermal conditions including pasteurization and thermization conditions. Efficient inactivation of the virus was observed in all tested conditions, except for thermization at 50C 10 min . Utilizing a submerged coil system with temperature ramp up times that resemble commercial pasteurizers, we showed that the virus was rapidly inactivated by pasteurization and most thermization conditions . These results provide important insights on the food safety measures utilized in the dairy industry. S...

Background    In March 2024, the USDA confirmed the first case of highly pathogenic avian influenza (HPAI) spreading between dairy cattle herds in the United States.  This followed reports from dairy producers of an unusual illness in their lactating cows over the preceding 2-3 months.  Virus whole genome sequencing and modeling performed by USDA suggested a single spillover of HPAI H5N1, clade 2.3.4.4b, genotype B3.13 from wild birds into dairy cattle likely occurred between October 2023 and January 2024 (1).  Since then, federal, state, and industry partners have collaborated to address the HPAI threat in dairy cattle, resulting in two federal orders and the implementation of the National Milk Testing Strategy (NMTS).  States began enrolling in the NMTS in December 2024, in which they are continuing to conduct or now implementing state-wide bulk tank surveillance and/or milk processing plant silo monitoring.  Nevada was among the first to participate...

Abstract Following reports of HPAI H5N1 infections of dairy cattle in the United States (US) in March 2024, we established a Pan-Canadian Milk network to monitor retail milk in Canada. Milk samples from across Canada that had previously tested negative for influenza A virus (IAV) RNA were tested for the presence of anti-IAV nucleoprotein (NP) antibodies , as an indicator of past infection of dairy cattle. None of the 109 milk samples tested had evidence of anti-IAV NP antibodies. This is consistent with previous findings from our academic group as well as others including federal testing initiatives that have not found any IAV RNA in milk. Although not surprising given that no cases of H5N1 in cattle have been reported in Canada to date, this work further supports that the extensive outbreak in dairy cattle in the US has not extended northward into Canada, and the integrity of the Canadian milk supply remains intact. Source: MedRxIV,  https://www.medrxiv.org/content/10.1101/2025.01...

Abstract In late 2023 an H5N1 lineage of high pathogenicity avian influenza virus (HPAIV) began circulating in American dairy cattle.  Concerningly, high titres of virus were detected in cows’ milk , raising the concern that milk could be a route of human infection . Cows’ milk is typically pasteurised to render it safe for human consumption, but the effectiveness of pasteurisation on influenza viruses in milk was uncertain . To assess this, here we evaluate heat inactivation in milk for a panel of different influenza viruses. This includes human and avian influenza A viruses (IAVs), an influenza D virus that naturally infects cattle, and recombinant IAVs carrying contemporary avian or bovine H5N1 glycoproteins . At pasteurisation temperatures of 63 °C and 72 °C , we find that viral infectivity is rapidly lost and becomes undetectable before the times recommended for pasteurisation ( 30 minutes and 15 seconds, respectively ). We then show that an H5N1 HPAIV in milk is effectively i...