Inactivation of highly pathogenic avian influenza virus with high temperature short time continuous flow pasteurization and virus detection in bulk milk tanks.

Infections of dairy cattle with clade 2.3.4.4b H5N1 highly pathogenic avian influenza virus (HPAIV) were reported in March 2024 in the U.S. and viable virus was detected at high levels in raw milk from infected cows. This study aimed to determine the potential quantities of infectious HPAIV in raw milk in affected states where herds were confirmed positive by USDA for HPAIV (and therefore were not representative of the entire population), and to confirm that the commonly used continuous flow pasteurization using the FDA approved 72°C (161°F) for 15 s conditions for high temperature short time (HTST) processing, will inactivate the virus. Double-blinded raw milk samples from bulk storage tanks from farms (n=275) were collected in four affected states. Samples were screened for influenza A using quantitative real-time RT-PCR (qrRT-PCR) of which 158 (57.5%) were positive and were subsequently quantified in embryonating chicken eggs. Thirty-nine qrRT-PCR positive samples (24.8%) were positive for infectious virus with a mean titer of 3.5 log10 50% egg infectious doses (EID50) per mL. To closely simulate commercial milk pasteurization processing systems, a pilot-scale continuous flow pasteurizer was used to evaluate HPAIV inactivation in artificially contaminated raw milk using the most common legal conditions in the US: 72°C (161°F) for 15s. Among all replicates at two flow rates (n=5 at 0.5L/min; n=4 at 1L/min), no viable virus was detected. A mean reduction of ≥5.8 ± 0.2 log10 EID50/mL occurred during the heating phase where the milk is brought to 72.5°C before the holding tube. Estimates from heat-transfer analysis support that standard U.S. continuous flow HTST pasteurization parameters will inactivate >12 log10 EID50/mL of HPAIV, which is ∼9 log10 EID50/mL greater than the mean quantity of infectious virus detected in raw milk from bulk storage tank samples. These findings demonstrate that the US milk supply is safe when pasteurized.

Efficacy of commercial recombinant HVT vaccines against a North American clade 2.3.4.4b H5N1 highly pathogenic avian influenza virus in chickens.

The outbreak of clade 2.3.4.4b H5 highly pathogenic avian influenza (HPAI) in North America that started in 2021 has increased interest in applying vaccination as a strategy to help control and prevent the disease in poultry. Two commercially available vaccines based on the recombinant herpes virus of turkeys (rHVT) vector were tested against a recent North American clade 2.3.4.4b H5 HPAI virus isolate: A/turkey/Indiana/22-003707-003/2022 H5N1 in specific pathogen free white leghorn (WL) chickens and commercial broiler chickens. One rHVT-H5 vaccine encodes a hemagglutinin (HA) gene designed by the computationally optimized broadly reactive antigen method (COBRA-HVT vaccine). The other encodes an HA gene of a clade 2.2 virus (2.2-HVT vaccine). There was 100% survival of both chicken types COBRA-HVT vaccinated groups and in the 2.2-HVT vaccinated groups there was 94.8% and 90% survival of the WL and broilers respectively. Compared to the 2.2-HVT vaccinated groups, WL in the COBRA-HVT vaccinated group shed significantly lower mean viral titers by the cloacal route and broilers shed significantly lower titers by the oropharyngeal route than broilers. Virus titers detected in oral and cloacal swabs were otherwise similar among both vaccine groups and chicken types. To assess antibody-based tests to identify birds that have been infected after vaccination (DIVA-VI), sera collected after the challenge were tested with enzyme-linked lectin assay-neuraminidase inhibition (ELLA-NI) for N1 neuraminidase antibody detection and by commercial ELISA for detection of antibodies to the NP protein. As early as 7 days post challenge (DPC) 100% of the chickens were positive by ELLA-NI. ELISA was less sensitive with a maximum of 75% positive at 10DPC in broilers vaccinated with 2.2-HVT. Both vaccines provided protection from challenge to both types of chickens and ELLA-NI was sensitive at identifying antibodies to the challenge virus therefore should be evaluated further for DIVA-VI.

Inactivation of Avian Influenza Virus Inoculated into Ground Beef Patties Cooked on a Commercial Open-Flame Gas Grill.

With the emergence of clade 2.3.4.4b H5N1 highly pathogenic avian influenza virus (AIV) infection of dairy cattle and its subsequent detection in raw milk, coupled with recent AIV infections affecting dairy farm workers, experiments were conducted to affirm the safety of cooked ground beef related to AIV because such meat is often derived from cull dairy cows. Specifically, retail ground beef (percent lean:fat = ca. 80:20) was inoculated with a low pathogenic AIV (LPAIV) isolate to an initial level of 5.6 log10 50% egg infectious doses (EID50)  per 300 g patty. The inoculated meat was pressed into patties (ca. 2.54 cm thick, ca. 300 g each) and then held at 4 °C for up to 60 min. In each of the two trials, two patties for each of the following three treatments were cooked on a commercial open-flame gas grill to internal instantaneous temperatures of 48.9 °C (120°F), 62.8 °C (145°F), or 71.1 °C (160°F), but without any dwell time. Cooking inoculated ground beef patties to 48.9 °C (ave. cooking time of ca. 15 min) resulted in a mean reduction of ≥2.5 ± 0.9 log10 EID50 per 300 g of ground beef as assessed via quantification of virus in embryonating chicken eggs (ECEs). Likewise, cooking patties on a gas grill to 62.8 °C (ave. cooking time of ca. 21 min) or to the USDA FSIS recommended minimum internal temperature for ground beef of 71.1 °C (ave. cooking time of ca. 24 min) resulted in a reduction to nondetectable levels from initial levels of ≥5.6 log10 EID50 per 300 g. These data establish that levels of infectious AIV are substantially reduced within inoculated ground beef patties (20% fat) using recommended cooking procedures.

Introduction of Avian metapneumovirus subtype A to the United States: molecular insights and implications.

Avian metapneumovirus (aMPV) poses a significant threat to the poultry industry worldwide, primarily affecting turkeys and chickens. The recent detection of aMPV-A and -B subtypes in the United States marks a significant shift after a prolonged period free of aMPV following the eradication of the previously circulating subtype C. Hence, the demand for molecular diagnostic tests for aMPV has arisen due to their limited availability in the US market. In this study, we present the molecular characterization based on the complete genome sequence of aMPV subtype A, which was detected in the US for the first time. Four RT-qPCR positive samples were subjected to next-generation sequencing analysis, resulting in the assembly of one complete and one near-complete genome sequences. Phylogenetic analysis revealed that the isolated strains clustered within the aMPV-A subtype and were most closely related to recent Mexican strains. A detailed amino acid analysis identified unique mutations in the G gene of the US isolates compared to Mexican strains. Additionally, we compared the performance, cross-reactivity, and limit of detection of our revised aMPV subtype-specific RT-qPCR test with two commercial kits, demonstrating similar detection and subtyping capabilities. These findings highlight the importance of accurate diagnostic methods for disease management in the poultry industry, provide valuable insights into the epidemiology of aMPV, and underscore the need for continued vigilance and surveillance to mitigate its impact on poultry production.

Characterization of highly pathogenic avian influenza virus in retail dairy products in the US.

In March 2024, clade 2.3.4.4b H5N1 highly pathogenic avian influenza virus (HPAIV) was detected in dairy cattle in the US, and it was discovered that the virus could be detected in raw milk. Although affected cow's milk is diverted from human consumption and current pasteurization requirements are expected to reduce or eliminate infectious HPAIV from the milk supply, a study was conducted to characterize whether the virus could be detected by quantitative real-time RT-PCR (qrRT-PCR) in pasteurized retail dairy products and, if detected, to determine whether the virus was viable. From 18 April to 22 April 2024, a total of 297 samples of Grade A pasteurized retail milk products (23 product types) were collected from 17 US states that represented products from 132 processors in 38 states. Viral RNA was detected in 60 samples (20.2%), with qrRT-PCR-based quantity estimates (non-infectious) of up to 5.4log1050% egg infectious doses per mL, with a mean and median of 3.0log10/mL and 2.9log10/mL, respectively. Samples that were positive for type A influenza by qrRT-PCR were confirmed to be clade 2.3.4.4 H5 HPAIV by qrRT-PCR. No infectious virus was detected in any of the qrRT-PCR-positive samples in embryonating chicken eggs. Further studies are needed to monitor the milk supply, but these results provide evidence that the infectious virus did not enter the US pasteurized milk supply before control measures for HPAIV were implemented in dairy cattle.IMPORTANCEHighly pathogenic avian influenza virus (HPAIV) infections in US dairy cattle were first confirmed in March 2024. Because the virus could be detected in raw milk, a study was conducted to determine whether it had entered the retail food supply. Pasteurized dairy products were collected from 17 states in April 2024. Viral RNA was detected in one in five samples, but infectious virus was not detected. This provides a snapshot of HPAIV in milk products early in the event and reinforces that with current safety measures, infectious viruses in milk are unlikely to enter the food supply.

Spillover of highly pathogenic avian influenza H5N1 virus to dairy cattle.

Highly pathogenic avian influenza (HPAI) H5N1 clade 2.3.4.4b virus has caused the death of millions of domestic birds and thousands of wild birds in the U.S. since January, 20221-4 Throughout this outbreak, spillovers to mammals have been frequently documented5-12. We report spillover of HPAI H5N1 virus in dairy cattle herds across several states in the U.S. The affected cows displayed clinical signs encompassing decreased feed intake, altered fecal consistency, respiratory distress, and decreased milk production with abnormal milk. Infectious virus and viral RNA were consistently detected in milk from affected cows. Viral distribution in tissues via immunohistochemistry and in situ hybridization revealed a distinct tropism of the virus for the epithelial cells lining the alveoli of the mammary gland in cows. Whole viral genome sequences recovered from dairy cows, birds, domestic cats, and a raccoon from affected farms indicated multidirectional interspecies transmissions. Epidemiologic and genomic data revealed efficient cow-to-cow transmission after apparently healthy cows from an affected farm were transported to a premise in a different state. These results demonstrate the transmission of HPAI H5N1 clade 2.3.4.4b virus at a non-traditional interface underscoring the ability of the virus to cross species barriers.

Unraveling frontiers in poultry health (part 1) - Mitigating economically important viral and bacterial diseases in commercial Chicken and Turkey production.

This symposium offered up-to-date perspectives on field experiences and the latest research on significant viral and bacterial diseases affecting poultry. A highlight was the discussion on the use of enteroids as advanced in vitro models for exploring disease pathogenesis. Outcomes of this symposium included identifying the urgent need to improve the prevention and control of avian influenza by focusing research on vaccine effectiveness. In this regard, efforts should focus on enhancing the relatedness of vaccine antigen to the field (challenge) virus strain and improving immunogenicity. It was also revealed that gangrenous dermatitis could be controlled through withholding or restricting the administration of ionophores during broiler life cycle, and that administration of microscopic polymer beads (gel) based-live coccidia vaccines to chicks could be used to reduce necrotic enteritis-induced mortality. It was emphasized that effective diagnosis of re-emerging Turkey diseases (such as blackhead, fowl cholera, and coccidiosis) and emerging Turkey diseases such as reoviral hepatitis, reoviral arthritis, Ornithobacterium rhinotracheale infection, and strepticemia require complementarity between investigative research approaches and production Veterinarian field approaches. Lastly, it was determined that the development of a variety of functionally-specific enteroids would expedite the delineation of enteric pathogen mechanisms and the identification of novel vaccine adjuvants.

The 28S rRNA RT-qPCR assay for host depletion evaluation to enhance avian virus detection in Illumina and Nanopore sequencing.

Abundant host and bacterial sequences can obscure the detection of less prevalent viruses in untargeted next-generation sequencing (NGS). Efficient removal of these non-targeted sequences is vital for accurate viral detection. This study presents a novel 28S ribosomal RNA (rRNA) RT-qPCR assay designed to assess the efficiency of avian rRNA depletion before conducting costly NGS for the detection of avian RNA viruses. The comprehensive evaluation of this 28S-test focuses on substituting DNase I with alternative DNases in our established depletion protocols and finetuning essential parameters for reliable host rRNA depletion. To validate the effectiveness of the 28S-test, we compared its performance with NGS results obtained from both Illumina and Nanopore sequencing platforms. This evaluation utilized swab samples from chickens infected with highly pathogenic avian influenza virus, subjected to established and modified depletion protocols. Both methods significantly reduced host rRNA levels, but using the alternative DNase had superior performance. Additionally, utilizing the 28S-test, we explored cost- and time-effective strategies, such as reduced probe concentrations and other alternative DNase usage, assessed the impact of filtration pre-treatment, and evaluated various experimental parameters to further optimize the depletion protocol. Our findings underscore the value of the 28S-test in optimizing depletion methods for advancing improvements in avian disease research through NGS.

Efficacy of recombinant H5 vaccines delivered in ovo or day of age in commercial broilers against the 2015 U.S. H5N2 clade 2.3.4.4c highly pathogenic avian Influenza virus.

BACKGROUND: Avian influenza is a highly contagious, agriculturally relevant disease that can severely affect the poultry industry and food supply. Eurasian-origin H5Nx highly pathogenic avian influenza viruses (HPAIV) (clade 2.3.4.4) have been circulating globally in wild birds with spill over into commercial poultry operations. The negative impact to commercial poultry renewed interest in the development of vaccines against these viruses to control outbreaks in the U.S. METHODS: The efficacy of three recombinant H5 vaccines delivered in ovo or day of age were evaluated in commercial broilers challenged with the 2015 U.S. H5N2 clade 2.3.4.4c HPAIV. The recombinant vaccines included an alphavirus RNA particle vaccine (RP-H5), an inactivated reverse genetics-derived (RG-H5) and recombinant HVT vaccine (rHVT-AI) expressing H5 hemagglutinin (HA) genes. In the first experiment, in ovo vaccination with RP-H5 or rHVT-AI was tested against HPAI challenge at 3 or 6 weeks of age. In a second experiment, broilers were vaccinated at 1 day of age with a dose of either 107 or 108 RP-H5, or RG-H5 (512 HA units (HAU) per dose). RESULTS: In experiment one, the RP-H5 provided no protection following in ovo application, and shedding titers were similar to sham vaccinated birds. However, when the RP-H5 was delivered in ovo with a boost at 3 weeks, 95% protection was demonstrated at 6 weeks of age. The rHVT-AI vaccine demonstrated 95 and 100% protection at 3 and 6 weeks of age, respectively, of challenged broilers with reduced virus shedding compared to sham vaccinated birds. Finally, when the RP-H5 and rHVT vaccines were co-administered at one day of age, 95% protection was demonstrated with challenge at either 3 or 6 weeks age. In the second experiment, the highest protection (92%) was observed in the 108 RP-H5 vaccinated group. Significant reductions (p < 0.05) in virus shedding were observed in groups of vaccinated birds that were protected from challenge. The RG-H5 provided 62% protection from challenge. In all groups of surviving birds, antibody titers increased following challenge. CONCLUSIONS: Overall, these results demonstrated several strategies that could be considered to protected broiler chickens during a H5 HPAI challenge.