Low Pathogenic Avian Influenza Virus Research Articles

Reassortants of the Highly Pathogenic Influenza Virus A/H5N1 Causing Mass Swan Mortality in Kazakhstan from 2023 to 2024

In the winter of 2023/2024, the mass death of swans was observed on Lake Karakol on the eastern coast of the Caspian Sea. From 21 December 2023 to 25 January 2024, 1132 swan corpses (Cygnus olor, Cygnus cygnus) were collected and disposed of on the coast by veterinary services and ecologists. Biological samples were collected from 18 birds for analysis at different dates of the epizootic. It was found that the influenza outbreak was associated with a high concentration of migrating birds at Lake Karakol as a result of a sharp cold snap in the northern regions. At different dates of the epizootic, three avian influenza A/H5N1 viruses of clade 2.3.4.4.b were isolated from dead birds and identified as highly pathogenic viruses (HPAIs) based on the amino acid sequence of the hemagglutinin multi-base proteolytic cleavage site (PLREKRRRKR/G). A phylogenetic analysis showed that the viruses isolated from the swans had reassortations in the PB2, PB1, and NP genes between highly pathogenic (HP) and low-pathogenic (LP) avian influenza viruses. Avian influenza viruses A/Cygnus cygnus/Karakol lake/01/2024(H5N1) and A/Mute swan/Karakol lake/02/2024(H5N1) isolated on 10 January 2024 received PB2, PB1, and NP from LPAIV, while A/Mute swan/Mangystau/9809/2023(H5N1) isolated on 26 December 2023 received PB1 and NP from LPAIV, indicating that the H5N1 viruses in this study are new reassortants. All viruses showed amino acid substitutions in the PB2, PB1, NP, and NS1 segments, which are critical for enhanced virulence or adaptation in mammals. An analysis of the genomes of the isolated viruses showed that bird deaths during different periods of the epizootic were caused by different reassortant viruses. Kazakhstan is located at the crossroads of several migratory routes of migratory birds, and the possible participation of wild birds in the introduction of various pathogens into the regions of Kazakhstan requires further study.

Transmission dynamics of highly pathogenic avian influenza virus at the wildlife-poultry-environmental interface: A case study

Avian influenza viruses (AIVs) regularly circulate between wild and domestic bird populations. Following several high-profile outbreaks, highly pathogenic AIVs (HPAIV) with zoonotic potential have been the subject of increasing attention. While we know that HPAIV is transmitted between domestic birds, wildlife, and the environment, little is known about persistence and spillover/back at these interfaces. We integrated the test results of samples collected on and around an infected domestic poultry premise (IP) where H5N1 HPAIV was confirmed in a flock of poultry in 2022 in Southern Ontario, Canada to explore the transmission cycle of AIVs in wildlife and the environment. We sampled a captive flock of Mallards (Anas platyrhynchos) that resided on site, sediment samples collected from water bodies on site, and examined samples collected through surveillance within a 100 km radius of the IP from live wild ducks and sick and dead wildlife. We found serologic evidence of H5 exposure in the captive mallards that resided on site despite no evidence of morbidity or mortality in these birds and no PCR positive detections from samples collected at two different timepoints. Genetic material from the same H5N1 HPAIV subtype circulating in the domestic birds and from low pathogenicity avian influenza viruses were detected in wetlands on site. The results of live and sick and dead surveillance conducted within a 100 km radius confirmed that the virus was circulating in wildlife before and after IP confirmation. These results suggest that biosecurity remains the most critical aspect of minimising spillover/back risk in a virus that has been shown to circulate in asymptomatic wild birds and persist in the surrounding environment.

Comparative analysis of innate immune responses in Sonali and broiler chickens infected with tribasic H9N2 low pathogenic avian influenza virus

BackgroundH9N2 avian influenza viruses have been circulating in Bangladesh since 2006, affecting multiple avian species and resulting in economic losses. The recent emergence of tribasic strains, along with co-infections, has increased the risk to poultry health. Therefore, the study aimed to compare the immune responses of Sonali (crossbred) and commercial broiler chickens infected with tribasic H9N2 low pathogenic avian influenza (LPAI) virus.MethodsFollowing H9N2 infection, proinflammatory (IL-6, IL-8, IL-1β and TNF-α) and antiviral (IFN-β and IFN-γ) cytokine expressions were observed in the trachea, lungs, intestine, and lymphoid tissues in Sonali and broiler chickens from 1 day post infection (dpi) to 10 dpi by qPCR.ResultsSonali chickens exhibited significantly higher proinflammatory and antiviral cytokine expressions in the trachea at 3–7 days post infection (dpi), while broiler chickens showed lower immune responses. Broiler chickens displayed prolonged IL-6, IL-8, and IL-1β expression in lungs at 3–10 dpi compared to Sonali chickens. In the intestine, broiler chickens showed higher IL-6 and IL-8 expression that peaks at 1–3 dpi, while in Sonali chickens only IL-1β elevated at 10 dpi. In response to the H9N2 viruses, broiler chickens exhibited a stronger early IFN-β responses and a delayed IFN-γ responses in their lymphoid organs compared to Sonali chickens.ConclusionThis suggests distinct immune profiles between the chicken types in response to the H9N2 infection. The information sheds light on the function of innate immunity in the pathophysiology of currently circulating tribasic H9N2 virus and could assist in effective controlling of avian influenza virus spread in poultry and designing vaccines.

Surveillance for Avian Influenza in Wild Birds in the Lombardy Region (Italy) in the Period 2022–2024

Influenza A virus (AIV) circulation was investigated in the Lombardy region, during 2022–2024, in wild ducks (through hunting and sampling of faecal samples within natural parks) and wild birds found dead. Samples were analysed through real-time RT-PCRs for Influenza A virus, H5 and H7. Whole genome sequencing was performed on AIV-positive samples. Screening of 3497 hunted Anatidae revealed a total of 184 positive samples. Complete sequencing of 136 samples highlighted the presence of 21 different subtypes ranging from H1N1 to H12N5. The H5N1 HPAIV (high pathogenic AIV) subtype, clade 2.3.4.4b, was the most common during the 2022–2023 winter season (31.8%), while H5 LPAI (low pathogenic AIV) strains were the most prevalent (28.6%) in the 2023–2024 season. The molecular survey on wild birds found dead (n = 481) showed two positive buzzards (14%, 2/14), one grey heron (5.5%, 1/18) and one kestrel (7.6%, 1/13). Regarding the order of Charadriiformes, the dead gulls sampled in 2022 (17 birds) were all negative, whereas 85 out of 167 (51%) individuals were positive in 2023. All positives were caused by an H5N1 HPAIV clade 2.3.4.4b virus belonging to genotype BB. All the faecal samples (1699) received from passive surveillance in nature parks were analysed for AIV with negative results.

Survivability of low pathogenic avian influenza virus in aqueous poultry manure fertilizer

Avian influenza has been a growing problem in the United States for decades. Low pathogenic avian influenza (LPAI) virus causes low morbidity, while highly pathogenic avian influenza (HPAI) has a devastating effect on the poultry industry causing high morbidity and mortality worldwide. Manure from large and small poultry operations is spread on crop fields as a fertilizer. During an outbreak of avian influenza in a farm it is critical to adequately process the manure to kill the virus before its use in agriculture. Aqueous extraction of poultry manure at high temperature (55-65°C) is an attractive method for treatment of poultry manure, which extracts the nutrients and kills harmful microbes from the manure. The end product is a nutrient rich liquid that can be safely applied to fields or used in hydroponic operations, with nutrients that are more bioavailable to the plant compared to dry litter. However, we do not know whether aqueous high temperature extraction processes completely kill avian influenza virus present in the manure. Our aim in this study was to determine the competence of the aqueous extraction of poultry manure on avian influenza virus inactivation. We optimized the procedure using a LPAI virus isolate in a biosafety level 2 cabinet. Our data indicated that LPAI virus including the hemagglutinin protein of the virus were completely destroyed when the poultry manure was processed by aqueous extraction at temperatures ≥ 55°C for one hour. Future research will identify the conditions required for inactivating HPAI virus using this optimized procedure by performing the study in a BSL3 facility.

Contrasting dynamics of two incursions of low-pathogenicity avian influenza virus into Australia.

The current panzootic of high pathogenicity avian influenza virus H5N1 demonstrates how viral incursions can have major ramifications for wildlife and domestic animals. Herein, we describe the recent incursion into Australia of two low pathogenicity avian influenza virus subtypes, H4 and H10, that exhibited contrasting evolutionary dynamics. Viruses detected from national surveillance and disease investigations between 2020 and 2022 revealed 27 genomes, 24 of which have at least one segment more closely related to Eurasian or North American avian influenza lineages than those already circulating in Australia. Phylogenetic analysis revealed that H4 viruses circulating in shorebirds represent a recent incursion from Asia that is distinct from those circulating concurrently in Australian waterfowl. Analysis of the internal segments further demonstrates exclusive, persistent circulation in shorebirds. This contrasts with H10, where a novel lineage has emerged in wild waterfowl, poultry, and captive birds across Australia and has likely replaced previously circulating H10 lineages through competitive exclusion. Elucidating different dynamics for avian influenza incursions supports effective disease risk identification and communication that better informs disease preparedness and response.

Preparation and Antigenic Site Identification of Monoclonal Antibodies against PB1 Protein of H9N2 Subtype AIV.

Recently, low pathogenic avian influenza virus (LPAIV), including H9N2 subtype, has been common clinical epidemic strains, and is widely distributed globally. The PB1 protein is a key component of the viral RNA polymerase complex (vRNP), and is vital to viral transcription and translation. In this study, to investigate the antigenic determinants in the PB1 protein, the truncated PB1 sequence (1bp-735bp) from H9N2 subtype AIV was amplified with PCR, and expressed in plasmid pET-28a (+). After purification, the recombinant PB1 protein was used to immunize BALB/c mice. Following immunization, hybridoma cells producing PB1-specific monoclonal antibodies were generated through the fusion of splenic lymphocytes with SP2/0 cells. Then, four stable hybridoma cell lines (5F12, 5B3, 2H9, and 3E6) were screened using indirect ELISA and Western blotting. Furthermore, two antigenic sites, 67NPIDGPLPED76 and 97ESHPGIFENS106, were identified through the construction of truncated overlapping fragments of the PB1 protein. These sites were conserved among 28 AIV strains, and were located on the PB1 protein surface. The findings offer a theoretical reference for the development and improvement of H9N2 vaccines and offer biological materials for virus detection during AIV infection mechanisms.

M2e nanovaccines supplemented with recombinant hemagglutinin protect chickens against heterologous HPAI H5N1 challenge

Current poultry vaccines against influenza A viruses target the globular head region of the hemagglutinin (HA1), providing limited protection against antigenically divergent strains. Experimental subunit vaccines based on the conserved ectodomain of the matrix protein 2 (M2e) induce cross-reactive antibody responses, but fail to fully prevent virus shedding after low pathogenic avian influenza (LPAI) virus challenge, and are ineffective against highly pathogenic avian influenza (HPAI) viruses. This study assessed the benefits of combining nanoparticles bearing three tandem M2e repeats (NR-3M2e nanorings or NF-3M2e nanofilaments) with an HA1 subunit vaccine in protecting chickens against a heterologous HPAI H5N1 virus challenge. Chickens vaccinated with the combined formulations developed M2e and HA1-specific antibodies, were fully protected from clinical disease and mortality, and showed no histopathological lesions or virus shedding, unlike those given only HA1, NR-3M2e, or NF-3M2e. Thus, the combined vaccine formulations provided complete cross-protection against HPAI H5N1 virus, and prevented environmental virus shedding, crucial for controlling avian influenza outbreaks.

Financial impact of low pathogenic avian influenza virus subtype H9N2 on commercial broiler chicken and egg layer production systems in Pakistan

Low Pathogenic Avian Influenza (LPAI) subtype H9N2 is endemic in Pakistan and impacts poultry farming through disease related mortality, poor weight gain and reduced egg production. This study aims to estimate the farm-level financial impact of LPAI H9N2 infection on commercial broiler and layer production systems in Pakistan.A questionnaire based cross-sectional survey of 138 broiler farms and 136 layer farms in Pakistan was conducted in 2019. Primary data collected by cross-sectional survey along with expert opinion and published literature were used to parameterize five stochastic production and gross margin models for three broiler and two layer production systems: fully integrated production (FIP), partially integrated production (PIP) and independent farming production (IP) systems. Partial budget analysis were then carried out to estimate the financial impact of LPAI H9N2.Results indicate that in broiler production systems, starting with 35,000 day old chicks (DOC) per batch, the net cost of disease (million PKR/production cycle) was estimated at 4.10 (14,862 USD), 4.62 (16,747 USD) and 2.46 (8917 USD) for IP, PIP and FIP systems, respectively. The disease produced a negative gross margin (defined here as revenue minus replacement and variable costs) in IP (-53 PKR (-0.19 USD)/DOC bought) and PI (-25 PKR (-0.091 USD)/DOC bought) systems, while remained positive for FIP systems (87 PKR (0.32 USD)/DOC bought). For layer production systems, (mean flock size as 48,000 DOCs) the net cost (million PKR/production cycle) was 29.75 (107,095.21 USD) and 29.51 (106,223.45 USD) IP and PIP systems, respectively, and produced negative gross margin in both systems.The outcomes of the study highlight the vulnerability of independent and partially integrated production systems to the disease. These findings also offer a decision-making tool to the farmers and policy makers to evaluate avian influenza surveillance systems and control interventions in Pakistan.

PB1-F2 of low pathogenicity H7N7 restricts apoptosis in avian cells

Avian influenza viruses (AIV) pose a continuous challenge to global health and economy. While countermeasures exist to control outbreaks in poultry, the persistent circulation of AIV in wild aquatic and shorebirds presents a significant challenge to effective disease prevention efforts. PB1-F2 is a non-structural protein expressed from a second open reading frame (+1) of the polymerase basic 1 (PB1) segment. The sequence and length of the PB1-F2 protein can vary depending on the host of origin. While avian isolates typically carry full-length PB1-F2, isolates from mammals, often express truncated forms. The selective advantage of the full-length PB1-F2 in avian isolates is not fully understood. Most research on the role of PB1-F2 in influenza virus replication has been conducted in mammalian systems, where PB1-F2 interfered with the host immune response and induced apoptosis. Here, we used Low Pathogenicity (LP) AIV H7N7 expressing full-length PB1-F2 as well as a knockout mutant. We found that the full-length PB1-F2 of LPAIV prolonged survival of infected cells by limiting apoptotic cell death. Furthermore, PB1-F2 knockout LPAIV significantly decreased MHC-I expression on fibroblasts, delayed tissue healing and increased phagocytic uptake of infected cells, whereas LPAIV expressing PB1-F2 has limited effects. These findings indicate that full-length PB1-F2 enables AIV to cause prolonged infections without severely harming the avian host. Our observations may explain maintenance of AIV in the natural bird reservoir in absence of severe clinical signs.

Activation of Antiviral Host Responses against Avian Influenza Virus and Remodeling of Gut Microbiota by rLAB Vector Expressing rIL-17A in Chickens.

Low-pathogenic avian influenza virus (LPAIV) remains the most common subtype of type-A influenza virus that causes moderate to severe infection in poultry with significant zoonotic and pandemic potential. Due to high mutability, increasing drug resistance, and limited vaccine availability, the conventional means to prevent intra- or interspecies transmission of AIV is highly challenging. As an alternative to control AIV infections, cytokine-based approaches to augment antiviral host defense have gained significant attention. However, the selective application of cytokines is critical since unregulated expression of cytokines, particularly proinflammatory ones, can cause substantial tissue damage during acute phases of immune responses. Moreover, depending on the type of cytokine and its impact on intestinal microbiota, outcomes of cytokine-gut microflora interaction can have a critical effect on overall host defense against AIV infections. Our recent study demonstrated some prominent roles of chicken IL-17A (ChIL-17A) in regulating antiviral host responses against AIV infection, however, in an in vitro model. For more detailed insights into ChIL-17A function, in the present study, we investigated whether ChIL-17A-meditated elevated antiviral host responses can translate into effective immune protection against AIV infection in an in vivo system. Moreover, considering the role of gut health in fostering innate or local host responses, we further studied the contributory relationships between gut microbiota and host immunity against AIV infection in chickens. For this, we employed a recombinant lactic acid-producing bacterial (LAB) vector, Lactococcus lactis, expressing ChIL-17A and analyzed the in vivo functionality in chickens against an LPAIV (A/H9N2) infection. Our study delineates that mucosal delivery of rL. lactis expressing ChIL-17A triggers proinflammatory signaling cascades and can drive a positive shift in phylum Firmicutes, along with a marked decline in phylum Actinobacteriota and Proteobacteria, favoring effective antiviral host responses against AIV infection in chickens. We propose that ChIL-17A-mediated selective expansion of beneficial gut microbiota might form a healthy microbial community that augments the effective immune protection against AIV infections in chickens.

Collaborative wildlife disease outbreak investigation and response at Bells Swamp Victoria, February 2023.

In February 2023, a report of morbidity and mortality in waterbirds triggered a collaborative regional wildlife disease outbreak investigation and response, led by Parks Victoria. Triage, rehabilitation and diagnosis of sick and dead birds were undertaken by Zoos Victoria (ZV), Agriculture Victoria, Vets for Compassion, Wildlife Victoria and Melbourne Veterinary School (MVS). The field response focused on collection of sick and dead birds for wildlife welfare, for diagnosis, and to reduce environmental contamination. Botulism was suspected, based on clinical signs and lack of significant gross pathology, and this diagnosis was confirmed by PCR testing. Low pathogenicity avian influenza (LPAI) viruses non H5 or H7 were detected in two birds and ruled out in all in others tested. These incidental, non-clinical LPAI detections are considered part of the natural wild bird virus community in Australia. A number of elements contributed to the collaborative effort. Regional individuals had the necessary connections for reporting, collecting and transporting birds. There was rapid determination by the Victorian Department of Energy, Environment and Climate Action (DEECA) that Parks Victoria, as the land managers, should lead the response. Zoos Victoria provided capacity and expertise in wildlife triage and rehabilitation, and Agriculture Victoria, ZV and MVS were responsible for veterinary management of the response and diagnosis. Field investigation and response were conducted by Parks Victoria, Agriculture Victoria, MVS and veterinary teams from Vets for Compassion and Wildlife Victoria. Wildlife Health Australia (WHA) provided guidance and information, approved National Significant Disease Investigation Program funding and captured the event in the national wildlife health information database. Communication and media were important for community understanding of the event.

High and low pathogenicity avian influenza virus discrimination and prediction based on volatile organic compounds signature by SIFT-MS: a proof-of-concept study

High and low pathogenicity avian influenza viruses (HPAIV, LPAIV) are the primary causes of poultry diseases worldwide. HPAIV and LPAIV constitute a major threat to the global poultry industry. Therefore, early detection and well-adapted surveillance strategies are of the utmost importance to control the spread of these viruses. Volatile Organic Compounds (VOCs) released from living organisms have been investigated over the last decades as a diagnostic strategy. Mass spectrometry instruments can analyze VOCs emitted upon viral infection. Selected ion flow tube mass spectrometry (SIFT-MS) enables direct analysis of cell headspace in less than 20 min. As a proof-of-concept study, we investigated the ability of a SIFT-MS coupled sparse Partial Least Square-Discriminant Analysis analytical workflow to discriminate IAV-infected cells. Supernatants of HPAIV, LPAIV, and control cells were collected from 1 to 72 h post-infection and analyzed using our analytical workflow. At each collection point, VOCs' signatures were first identified based on four independent experiments and then used to discriminate the infectious status of external samples. Our results indicate that the identified VOCs signatures successfully discriminate, as early as 1-h post-infection, infected cells from the control cells and differentiated the HPAIV from the LPAIV infection. These results suggest a virus-dependent VOCs signature. Overall, the external samples' status was identified with 96.67% sensitivity, 100% specificity, and 97.78% general accuracy.

Evaluating the Impact of Low-Pathogenicity Avian Influenza H6N1 Outbreaks in United Kingdom and Republic of Ireland Poultry Farms during 2020.

In January 2020, increased mortality was reported in a small broiler breeder flock in County Fermanagh, Northern Ireland. Gross pathological findings included coelomitis, oophoritis, salpingitis, visceral gout, splenomegaly, and renomegaly. Clinical presentation included inappetence, pronounced diarrhoea, and increased egg deformation. These signs, in combination with increased mortality, triggered a notifiable avian disease investigation. High pathogenicity avian influenza virus (HPAIV) was not suspected, as mortality levels and clinical signs were not consistent with HPAIV. Laboratory investigation demonstrated the causative agent to be a low-pathogenicity avian influenza virus (LPAIV), subtype H6N1, resulting in an outbreak that affected 15 premises in Northern Ireland. The H6N1 virus was also associated with infection on 13 premises in the Republic of Ireland and six in Great Britain. The close genetic relationship between the viruses in Ireland and Northern Ireland suggested a direct causal link whereas those in Great Britain were associated with exposure to a common ancestral virus. Overall, this rapidly spreading outbreak required the culling of over 2 million birds across the United Kingdom and the Republic of Ireland to stamp out the incursion. This report demonstrates the importance of investigating LPAIV outbreaks promptly, given their substantial economic impacts.

In turkeys, unlike chickens, the non-structural NS1 protein does not play a significant role in the replication and tissue tropism of the H7N1 avian influenza virus

ABSTRACT The economic losses caused by high pathogenicity (HP) avian influenza viruses (AIV) in the poultry industry worldwide are enormous. Although chickens and turkeys are closely related Galliformes, turkeys are thought to be a bridging host for the adaptation of AIV from wild birds to poultry because of their high susceptibility to AIV infections. HPAIV evolve from low pathogenicity (LP) AIV after circulation in poultry through mutations in different viral proteins, including the non-structural protein (NS1), a major interferon (IFN) antagonist of AIV. At present, it is largely unknown whether the virulence determinants of HPAIV are the same in turkeys and chickens. Previously, we showed that mutations in the NS1 of HPAIV H7N1 significantly reduced viral replication in chickens in vitro and in vivo. Here, we investigated the effect of NS1 on the replication and virulence of HPAIV H7N1 in turkeys after inoculation with recombinant H7N1 carrying a naturally truncated wild-type NS1 (with 224 amino-acid “aa” in length) or an extended NS1 with 230-aa similar to the LP H7N1 ancestor. There were no significant differences in multiple-cycle viral replication or in the efficiency of NS1 in blocking IFN induction in the cell culture. Similarly, all viruses were highly virulent in turkeys and replicated at similar levels in various organs and swabs collected from the inoculated turkeys. These results suggest that NS1 does not play a role in the virulence or replication of HPAIV H7N1 in turkeys and further indicate that the genetic determinants of HPAIV differ in these two closely related galliform species.

The therapeutic efficacy of neem (Azadirecta indica) leaf extract against coinfection with Chlamydophila psittaci and low pathogenic avian influenza virus H9N2 in broiler chickens

Avian chlamydiosis is a serious avian infection that carries a significant zoonotic danger to the poultry industry. The respiratory co-infections caused by the low pathogenic avian influenza virus H9N2 (LPAIV H9N2) also cause significant financial losses in the poultry industry. The purpose of this study was to examine the pathogenicity of Chlamydophila psittaci, and LPAIV H9N2 individually and in combination in broiler chickens, as well as to determine whether or not aqueous neem (Azadirachta indica) leaf extract is effective against infections caused by these pathogens. Therefore, 120 broiler cobb chicks were equally divided into 4 groups (30 birds each) with triplicates with 10 birds. Broilers in group 1 (G1) were infected with only C. psittaci, broilers in group 2 (G2) were infected with only LPAIV H9N2, broilers in group 3 (G3) were infected with C. psittaci and LPAIV H9N2, and broilers in group 4 (G4) remained not challenged and non-treated with any therapeutic or preventive treatment (negative control). At 21 d postinfection (dpi), birds in G1, G2, and G3 were divided into 3 subgroups of 10 birds each: subgroup (A) remained infected and untreated (positive control), subgroup (B) infected and received oxytetracycline for 5 consecutive d, and subgroup (C) infected and received 8% aqueous neem leaf extract for 5 consecutive d. The multiplication of C. psittaci in birds in G1, in various tissues was evaluated using Giemsa staining and the data showed that multiplication was much higher in the lung, spleen, and liver from 6 h to 21 dpi, but low in the heart from 8 to 21 dpi. During simultaneous co-infection in G3, the birds developed significant clinical symptoms and postmortem lesions (PM). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect viral shedding from oropharyngeal and cloacal swabs between 2 dpi and 8 dpi, with cycle threshold (CT) values ranging from 22 to 24. In contrast, bacterial shedding began 6 h after infection and continued until 21 dpi, with CT values ranging from 23 to 26. Administration of an aqueous neem leaf extract at an 8% concentration (Group C) resulted in a numerical rise in average body weight across all treatment groups in the third and fourth week, as well as a reduction in LPAIV H9N2 and C. psittaci replication in the respiratory and gut of treated birds compared to those treated with oxytetracycline (Group B). Overall, respiratory co-infections pose a considerable risk to the poultry business, which is a big threat. To control C. psittaci and LPAIV H9N2 in broiler chickens, oral supplementation of 8% aqueous neem leaf extract is recommended. This treatment improves the birds' performance, as evidenced by an increase in their average body weight. In addition, the application of 8% aqueous neem leaf extract lowers C. psittaci replication within tissues and diminishes LPAIV H9N2 shedding.

Evaluating the epizootic and zoonotic threat of an H7N9 low-pathogenicity avian influenza virus (LPAIV) variant associated with enhanced pathogenicity in turkeys.

Between 2013 and 2017, the A/Anhui/1/13-lineage (H7N9) low-pathogenicity avian influenza virus (LPAIV) was epizootic in chickens in China, causing mild disease, with 616 fatal human cases. Despite poultry vaccination, H7N9 has not been eradicated. Previously, we demonstrated increased pathogenesis in turkeys infected with H7N9, correlating with the emergence of the L217Q (L226Q H3 numbering) polymorphism in the haemagglutinin (HA) protein. A Q217-containing virus also arose and is now dominant in China following vaccination. We compared infection and transmission of this Q217-containing 'turkey-adapted' (ty-ad) isolate alongside the H7N9 (L217) wild-type (wt) virus in different poultry species and investigated the zoonotic potential in the ferret model. Both wt and ty-ad viruses demonstrated similar shedding and transmission in turkeys and chickens. However, the ty-ad virus was significantly more pathogenic than the wt virus in turkeys but not in chickens, causing 100 and 33% mortality in turkeys respectively. Expanded tissue tropism was seen for the ty-ad virus in turkeys but not in chickens, yet the viral cell receptor distribution was broadly similar in the visceral organs of both species. The ty-ad virus required exogenous trypsin for in vitro replication yet had increased replication in primary avian cells. Replication was comparable in mammalian cells, and the ty-ad virus replicated successfully in ferrets. The L217Q polymorphism also affected antigenicity. Therefore, H7N9 infection in turkeys can generate novel variants with increased risk through altered pathogenicity and potential HA antigenic escape. These findings emphasize the requirement for enhanced surveillance and understanding of A/Anhui/1/13-lineage viruses and their risk to different species.

Red knots in Europe: a dead end host species or a new niche for highly pathogenic avian influenza?

The 2020/2021 epidemic in Europe of highly pathogenic avian influenza virus (HPAIV) of subtype H5 surpassed all previously recorded European outbreaks in size, genotype constellations and reassortment frequency and continued into 2022 and 2023. The causative 2.3.4.4b viral lineage proved to be highly proficient with respect to reassortment with cocirculating low pathogenic avian influenza viruses and seems to establish an endemic status in northern Europe. A specific HPAIV reassortant of the subtype H5N3 was detected almost exclusively in red knots (Calidris canutus islandica) in December 2020. It caused systemic and rapidly fatal disease leading to a singular and self-limiting mass mortality affecting about 3500 birds in the German Wadden Sea, roughly 1 % of the entire flyway population of islandica red knots. Phylogenetic analyses revealed that the H5N3 reassortant very likely had formed in red knots and remained confined to this species. While mechanisms of virus circulation in potential reservoir species, dynamics of spill-over and reassortment events and the roles of environmental virus sources remain to be identified, the year-round infection pressure poses severe threats to endangered avian species and prompts adaptation of habitat and species conservation practices.

Pathogen Surveillance in Swallows (family Hirundinidae): Investigation into Role as Avian Influenza Vector in Eastern Canada Agricultural Landscapes.

First detected in Atlantic Canada in December 2021, highly pathogenic avian influenza virus (HPAIV) subtype H5N1 clade 2.3.4.4b, A/Goose/Guangdong/1/96 lineage, has caused massive mortality in wild birds and domestic poultry in North America. Swallows (Hirundinidae), abundant in North American agricultural ecosystems, have been proposed as possible (bridge) species for HPAIV transmission between wild and domestic birds. We aimed to seek evidence of the potential role of swallows in bridging AIV infection between wild bird reservoirs and poultry flocks in eastern Canada. During a wide-scale outbreak of HPAIV in wild birds and poultry farms across eastern Canada, 200 samples were collected from swallow breeding sites in the Canadian provinces of New Brunswick, Nova Scotia, Ontario, and Quebec, June-August 2022. Samples came from Barn Swallow (Hirundo rustica; n=142), Tree Swallow (Tachycineta bicolor; n=56), and Cliff Swallow (Petrochelidon pyrrhonota; n=2) nests. All samples tested negative for AIV, suggesting that HPAIV and low pathogenic AIV (LPAIV) strains were probably not circulating widely in swallows during the 2022 breeding season in eastern Canada; thus swallows may present a low risk of transmitting AIV. Within a management context, these findings suggest that removing nests of Barn Swallows, a species at risk in Canada, from the exterior of biosecure domestic poultry facilities may not significantly reduce risks of HPAI transmission to poultry.

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.