Pathogenic Avian Influenza Virus Subtype Research Articles

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.

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.

Lack of Highly Pathogenic Avian Influenza H5N1 in the South Shetland Islands in Antarctica, Early 2023.

In January 2023, an active surveillance initiative was undertaken in the South Shetland Islands, Antarctica, with the specific objective of ascertaining evidence for the presence of avian influenza, and specifically the highly pathogenic avian influenza virus subtype H5N1 (HPAIV H5N1). The investigation encompassed diverse locations, including Hanna Point (Livingston Island), Lions Rump (King George Island), and Base Escudero (King George Island), with targeted observations on marine mammals (southern elephant seals), flying birds (the kelp gull, snowy sheathbill and brown skua), and penguins (the chinstrap penguin and gentoo penguin). The study encompassed the examination of these sites for signs of mass mortality events possibly attributable to HPAIV H5N1, as well as sampling for influenza detection by means of real-time RT-PCR. Two hundred and seven (207) samples were collected, including 73 fecal samples obtained from the environment from marine mammals (predominantly feces of southern elephant seals), and 77 cloacal samples from penguins of the genus Pygoscelis (predominantly from the gentoo penguin). No evidence of mass mortality attributable to HPAIV H5N1 was observed, and all the collected samples tested negative for the presence of the virus, strongly suggesting the absence of the virus in the Antarctic territory during the specified period. This empirical evidence holds significant implications for both the ecological integrity of the region and the potential zoonotic threats, underscoring the importance of continued surveillance and monitoring in the Antarctic ecosystem.

Genetic Characterization and Phylogeographic Analysis of the First H13N6 Avian Influenza Virus Isolated from Vega Gull in South Korea.

Avian influenza virus (AIV) is a pathogen with zoonotic and pandemic potential. Migratory birds are natural reservoirs of all known subtypes of AIVs, except for H17N10 and H18N11, and they have been implicated in previous highly pathogenic avian influenza outbreaks worldwide. This study identified and characterized the first isolate of the H13N6 subtype from a Vega gull (Larus vegae mongolicus) in South Korea. The amino acid sequence of hemagglutinin gene showed a low pathogenic AIV subtype and various amino acid substitutions were found in the sequence compared to the reference sequence and known H13 isolates. High sequence homology with other H13N6 isolates was found in HA, NA, PB1, and PA genes, but not for PB2, NP, M, and NS genes. Interestingly, various point amino acid mutations were found on all gene segments, and some are linked to an increased binding to human-type receptors, resistance to antivirals, and virulence. Evolutionary and phylogenetic analyses showed that all gene segments are gull-adapted, with a phylogeographic origin of mostly Eurasian, except for PB2, PA, and M. Findings from this study support the evidence that reassortment of AIVs continuously occurs in nature, and migratory birds are vital in the intercontinental spread of avian influenza viruses.

Highly pathogenic avian influenza virus H5N1 infection in skua and gulls in the United Kingdom, 2022.

The reemergence of the highly pathogenic avian influenza virus (HPAIV) subtype H5N1 in the United Kingdom in 2021-2022 has caused unprecedented epizootic events in wild birds and poultry. During the summer of 2022, there was a shift in virus transmission dynamics resulting in increased HPAIV infection in seabirds, and consequently, a profound impact on seabird populations. To understand the pathological impact of HPAIV in seabirds, we evaluated the virus antigen distribution and associated pathological changes in the tissues of great skua (Stercorarius skua, n = 8), long-tailed skua (Stercorarius longicaudus, n = 1), European herring gull (Larus argentatus, n = 5), and black-headed gull (Chroicocephalus ridibundus, n = 4), which succumbed to natural infection of HPAIV during the summer of 2022. Cases were collected from Shetland, including Scatness (mainland), No Ness (mainland), Clumlie (mainland), Hermaness (island), Fair Isle (island), Noss (island), and the West Midlands, South East, and South West of England. Grossly, gizzard ulceration was observed in one great skua and pancreatic necrosis was observed in 4 herring gulls, with intralesional viral antigen detected subsequently. Microscopical analysis revealed neuro-, pneumo-, lymphoid-, and cardiomyotropism of HPAIV H5N1, with the most common virus-associated pathological changes being pancreatic and splenic necrosis. Examination of the reproductive tract of the great skua revealed HPAIV-associated oophoritis and salpingitis, and virus replication within the oviductal epithelium. The emergence of HPAIV in seabirds Stercorariidae and Laridae, particularly during summer 2022, has challenged the dogma of HPAIV dynamics, posing a significant threat to wild bird life with potential implications for the reproductive performance of seabirds of conservation importance.

Concern regarding H3-subtype avian influenza virus.

The H3-subtype of avian influenza virus (AIV) is one of the most frequently detected low pathogenic avian influenza virus (LPAIV) subtypes in birds and fowls, causing substantial economic loss to the poultry industry. Most importantly, besides poultry, mammals could also be infected with it, such as swines, canines, equines, felines, and humans, posing a serious public health threat. This allows the virus to persist widely in poultry and wild birds for a long time, where it may mix with other subtypes, providing conditions for viral recombination or reassortment. Currently, the monitoring of H3-subtype AIV is inadequate, and there is a lack of effective prevention and control measures for H3-subtype AIV. Here, the epidemiology, phylogeny, and genetic variation of H3-subtype AIV were analyzed, and nonsynonymous and synonymous substitution rates (dN/dS) were calculated. Through these steps, we aimed to clarify the current epidemiological feature and evolutionary characteristics of H3-subtype AIV, and provide an operative reference for future scientific control of H3-subtype AIV.

Determinants for the presence of avian influenza virus in live bird markets in Bangladesh: Towards an easy fix of a looming one health issue

Highly pathogenic avian influenza virus subtype H5N1 endangers poultry, wildlife, and human health and is enzootic in large parts of Asia, with live bird markets (LBMs) as putative hotspots for their maintenance, amplification, and spread. To mitigate the extent of these and other avian influenza viruses (AIV) of concern, we aimed to increase our quantitative understanding of the factors determining the presence of avian influenza virus in LBM stalls. Between 2016 and 2017, we collected fecal or offal samples from 1008 stalls in 113 LBMs across the Dhaka and Rajshahi districts in Bangladesh. For each stall, samples were pooled and tested for the AIV matrix gene, followed by H5 and H9 subtyping using rRT-PCR. We detected Influenza A viral RNA in 49% of the stalls. Of the AIV positive samples, 52% and 24% were determined to be H5 and H9 viruses, respectively, which are both subtypes of considerable health concern. We used generalized linear mixed effect modelling to study AIV presence in individual stalls within LBMs as a function of 13 out of the 20 risk factors identified by FAO. We found that small and feasible improvements in cleaning and disinfection frequency, installing running water in stalls, and not mixing different breeds of chicken in the same cages had large impacts on the presence of AIV in stalls (Odds ratios 0.03–0.05). Next, cleaning vehicles used in poultry transport, not selling waterfowl with chickens in the same stall, buying stock directly from commercial farms, separating sick birds from healthy ones, and avoiding access by wild birds like house crows, also had major effects on lowering the risk of stalls having AIV (Odds ratios 0.16–0.33). These findings can be directly used in developing practical and affordable measures to reduce the prevalence of AIV in LBMs. Also, in settings with limited resources like Bangladesh, such mitigation may significantly contribute to reducing AIV circulation amongst poultry and spillover to wildlife and humans.

Avian sarcoma/leukosis virus (RCAS)-mediated over-expression of IFITM3 protects chicks from highly pathogenic avian influenza virus subtype H5N1

Broad-spectrum antiviral activities of interferon-induced transmembrane proteins (IFITMs) are primarily attributed to in vitro inhibition of viral entry. Here, we used an avian sarcoma-leukosis virus (RCAS)-based gene transfer system and successfully generated chicks that constitutively express chicken IFITM3 (chIFITM3). The chIFITM3-overexpressing chicks showed significant protection and disease tolerance against highly pathogenic avian influenza virus (HPAIV) H5N1 (Clade 2.2.1.2). The chicks, overexpressing chIFITM3, also showed delayed onset of clinical symptoms, reduced viral shedding, and alleviated histopathologic alterations compared to control and challenged chicks. These findings highlight that overexpression of chIFITM3 provide a substantial defense against zoonotic H5N1 in vivo.

Review on the Epizootiological Situation on Highly Pathogenic Avian Influenza around the World and in Russia in 2022

This paper describes the current situation on highly pathogenic avian influenza virus in 2022 and provides forecast of the possible further spread of avian influenza in Russia. In 2022, the circulation of a wide variety of highly pathogenic avian influenza virus subtypes, which have epizootiological and epidemiological significance, was recorded in the world. Outbreaks caused by highly pathogenic avian influenza virus were reported in over 60 countries. In addition, human infections with influenza viruses of the A(H5Nx) and A(H9N2) subtypes were registered. There was a large-scale epizootic which affected more than 10 regions of the European part of Russia and the Russian Far East in 2022. Outbreaks among wild birds and poultry were caused by the highly pathogenic influenza virus A(H5N1) of the clade 2.3.4.4b, at the same time genetic and antigenic diversity was observed among viruses circulating in Russia. Thus, an essential geographical role of the territory of Russia in the global spread of avian influenza virus has been shown once again, which highlights the importance of continuous avian influenza virus surveillance in the country.

Seroconversion of a Swine Herd in a Free-Range Rural Multi-Species Farm against HPAI H5N1 2.3.4.4b Clade Virus.

Starting from October 2021, several outbreaks of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 were reported in wild and domestic birds in Italy. Following the detection of an HPAIV in a free-ranging poultry farm in Ostia, province of Rome, despite the lack of clinical signs, additional virological and serological analyses were conducted on samples collected from free-ranging pigs, reared in the same holding, due to their direct contact with the infected poultry. While the swine nasal swabs were all RT-PCR negative for the influenza type A matrix (M) gene, the majority (%) of the tested pigs resulted serologically positive for the hemagglutination inhibition test and microneutralization assay, using an H5N1 strain considered to be homologous to the virus detected in the farm. These results provide further evidence of the worrisome replicative fitness that HPAI H5Nx viruses of the 2.3.4.4b clade have in mammalian species. Moreover, our report calls for additional active surveillance, to promptly intercept occasional spillover transmissions to domestic mammals in close contact with HPAI affected birds. Strengthened biosecurity measures and efficient separation should be prioritized in mixed-species farms in areas at risk of HPAI introduction.

Zoonotic Mutation of Highly Pathogenic Avian Influenza H5N1 Virus Identified in the Brain of Multiple Wild Carnivore Species.

Wild carnivore species infected with highly pathogenic avian influenza (HPAI) virus subtype H5N1 during the 2021-2022 outbreak in the Netherlands included red fox (Vulpes vulpes), polecat (Mustela putorius), otter (Lutra lutra), and badger (Meles meles). Most of the animals were submitted for testing because they showed neurological signs. In this study, the HPAI H5N1 virus was detected by PCR and/or immunohistochemistry in 11 animals and was primarily present in brain tissue, often associated with a (meningo) encephalitis in the cerebrum. In contrast, the virus was rarely detected in the respiratory tract and intestinal tract and associated lesions were minimal. Full genome sequencing followed by phylogenetic analysis demonstrated that these carnivore viruses were related to viruses detected in wild birds in the Netherlands. The carnivore viruses themselves were not closely related, and the infected carnivores did not cluster geographically, suggesting that they were infected separately. The mutation PB2-E627K was identified in most carnivore virus genomes, providing evidence for mammalian adaptation. This study showed that brain samples should be included in wild life surveillance programs for the reliable detection of the HPAI H5N1 virus in mammals. Surveillance of the wild carnivore population and notification to the Veterinary Authority are important from a one-heath perspective, and instrumental to pandemic preparedness.

Effect of Co-infection of Low Pathogenic Avian Influenza H9N2 Virus and Avian Pathogenic E. coli on H9N2-Vaccinated Commercial Broiler Chickens.

In the last 40 years, low pathogenic avian influenza virus (LPAIV) subtype H9N2 has been endemic in most Middle Eastern countries and of course Egypt which is one of the biggest poultry producers in the middle east region. The major losses with the H9N2 virus infections come from complicated infections in commercial broiler chickens, especially E. coli infection. In this work, 2,36,345 Arbor acres broiler chickens from the same breeder flock were placed equally in four pens, where two pens were vaccinated against LPAIV of subtype H9N2 virus, and the other two pens served as non-vaccinated controls. All were placed on the same farm under the same management conditions. A total of twenty birds from each pen were moved to biosafety level−3 chicken isolators (BSL-3) on days 21 and 28 of life and challenged with LPAIV-H9N2 or E. coli. Seroconversion for H9N2 was evaluated before and after the challenge. The recorded results revealed a significant decrease in clinical manifestations and virus shedding in terms of titers of shedding virus and number of shedders in vaccinated compared to non-vaccinated chickens. In groups early infected with LPAIV-H9N2 virus either vaccinated or not vaccinated, there was no significant difference in clinical sickness or mortalities in both groups, but in late infection groups with H9N2 alone, non-vaccinated infected group showed significantly higher clinical sickness in comparison with infected vaccinated group but also without mortality. In groups co-infected with E. coli (I/M) and H9N2, it showed 100% mortalities either in vaccinated or non-vaccinated H9N2 groups and thus reflect the high pathogenicity of used E. coli isolates, whereas in groups co-infected with E. coli (per os to mimic the natural route of infection) and LPAIV-H9N2, mortality rates were significantly higher in non-vaccinated groups than those vaccinated with H9N2 vaccine (15 vs. 5%). In conclusion, the use of the LPAIV H9N2 vaccine has significantly impacted the health status, amount of virus shed, and mortality of challenged commercial broilers, as it can minimize the losses and risks after co-infection with E. coli (orally) and LPAIV-H9N2 virus under similar natural route of infection in commercial broilers.

H5 low pathogenic avian influenza viruses maintained in wild birds in China

Low pathogenic avian influenza virus, H5 or H7 subtype, possesses the potential capability to change to highly pathogenic variant, which damages wild waterfowl, domestic poultry, and mammalian hosts. In regular active surveillance of avian influenza virus from wild birds in China in 2020, we isolated six H5 avian influenza viruses, including one H5N2, two H5N3, and three H5N8. Phylogenetic analysis indicated that the H5N2 and H5N3 isolates clustered into Eurasian lineage, whereas the H5N8 viruses were originated in North America. The HA proteins of six viruses carried the cleavage-site motif PQRETR↓GLF, which indicated low pathogenicity of the viruses in chickens. However, the N30D, I43M, and T215A mutations in M1 protein and the P42S, I106M, and C138F residues changed in NS1 protein, implying all viruses could exhibit increased virulence in mice. Viral replication kinetics in mammalian cells demonstrated that the three representative viruses had the ability to replicate in both MDCK cells and A549 cells with low titers. Even though two of three representatives, WS/SX/S3-620/2020(H5N3) and ML/AH/A3-770/2020(H5N8), did not replicate and transmit efficiently in poultry (chickens), they did replicate and transmit efficiently in waterfowl (ducks). Viral pathogenicity in mice indicated that both H5N2 and H5N3 viruses are able to replicate in the nasal turbinates and lungs of mice without prior adaptation, while the H5N8 virus could not. The intercontinental and cross-species transmission of viruses may continuously exist in China, thereby providing constant opportunities for virus reassortment with local resident AIVs. Thus, it is crucial to continuously monitor migration routes for AIVs by systematic surveillance.

Transgenic Chicks Expressing Interferon-Inducible Transmembrane Protein 1 (IFITM1) Restrict Highly Pathogenic H5N1 Influenza Viruses

Mammalian cells utilize a wide spectrum of pathways to antagonize the viral replication. These pathways are typically regulated by antiviral proteins and can be constitutively expressed but also exacerbated by interferon induction. A myriad of interferon-stimulated genes (ISGs) have been identified in mounting broad-spectrum antiviral responses. Members of the interferon-induced transmembrane (IFITM) family of proteins are unique among these ISGs due to their ability to prevent virus entry through the lipid bilayer into the cell. In the current study, we generated transgenic chickens that constitutively and stably expressed chicken IFITM1 (chIFITM1) using the avian sarcoma-leukosis virus (RCAS)-based gene transfer system. The challenged transgenic chicks with clinical dose 104 egg infective dose 50 (EID50) of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 (clade 2.2.1.2) showed 100% protection and significant infection tolerance. Although challenged transgenic chicks displayed 60% protection against challenge with the sub-lethal dose (EID50 105), the transgenic chicks showed delayed clinical symptoms, reduced virus shedding, and reduced histopathologic alterations compared to non-transgenic challenged control chickens. These finding indicate that the sterile defense against H5N1 HPAIV offered by the stable expression of chIFITM1 is inadequate; however, the clinical outcome can be substantially ameliorated. In conclusion, chIFITM proteins can inhibit influenza virus replication that can infect various host species and could be a crucial barrier against zoonotic infections.

Overview of the Epizootiological Situation on Highly Pathogenic Avian Influenza in Russia in 2020

This review describes the current situation on highly pathogenic avian influenza virus in 2020 and provides forecast of the possible further spread of avian influenza in Russia. In 2020, the circulation of a wide variety of highly pathogenic avian influenza virus subtypes which have epizootiological and epidemiological significance was recorded in the world. Outbreaks of highly pathogenic avian influenza were reported in over 30 countries. Apart from this, human infections with influenza viruses of the A/H5Nx and A/H9N2 subtypes were reported. There was a large-scale epizootic in Russia in 2020, which affected more than 10 regions. Outbreaks among wild birds and poultry were caused by the highly pathogenic influenza virus A/H5N8 of clade 2.3.4.4b. As a result of those outbreaks, more than 1.5 mil. poultry were killed or perished. It was revealed that strains of the influenza virus isolated in Russia have a high degree of identity with the strains circulating in Europe and Southeast Asia. Thus, it was shown again that the territory of Russia plays an important role in the global spread of avian influenza virus.

Virus Adaptation Following Experimental Infection of Chickens with a Domestic Duck Low Pathogenic Avian Influenza Isolate from the 2017 USA H7N9 Outbreak Identifies Polymorphic Mutations in Multiple Gene Segments.

In March 2017, highly pathogenic (HP) and low pathogenic (LP) avian influenza virus (AIV) subtype H7N9 were detected from poultry farms and backyard birds in several states in the southeast United States. Because interspecies transmission is a known mechanism for evolution of AIVs, we sought to characterize infection and transmission of a domestic duck-origin H7N9 LPAIV in chickens and genetically compare the viruses replicating in the chickens to the original H7N9 clinical field samples used as inoculum. The results of the experimental infection demonstrated virus replication and transmission in chickens, with overt clinical signs of disease and shedding through both oral and cloacal routes. Unexpectedly, higher levels of virus shedding were observed in some cloacal swabs. Next generation sequencing (NGS) analysis identified numerous non-synonymous mutations at the consensus level in the polymerase genes (i.e., PA, PB1, and PB2) and the hemagglutinin (HA) receptor binding site in viruses recovered from chickens, indicating possible virus adaptation in the new host. For comparison, NGS analysis of clinical samples obtained from duck specimen collected during the outbreak indicated three polymorphic sides in the M1 segment and a minor population of viruses carrying the D139N (21.4%) substitution in the NS1 segment. Interestingly, at consensus level, A/duck/Alabama (H7N9) had isoleucine at position 105 in NP protein, similar to HPAIV (H7N9) but not to LPAIV (H7N9) isolated from the same 2017 influenza outbreak in the US. Taken together, this work demonstrates that the H7N9 viruses could readily jump between avian species, which may have contributed to the evolution of the virus and its spread in the region.

Molecular Characterization, Pathogenesis and Immunological Effect of Avian Influenza H9N2 Virus Isolated From Broiler Chickens in Egypt During 2019

Many commercial flocks in Egypt were infected with the low pathogenic avian influenza virus (LPAIV) subtype H9N2, resulting in severe economic losses, particularly when combined with other viral and bacterial diseases. The current study aimed to investigate the genetic evolution of LPAIV (H9N2) in broiler farms in Egypt during 2019 and evaluate its effect on the viral pathogenesis, cytokine response, innate immunity and oxidative state in specific pathogen free (SPF) chicks. Sixty tracheal swabs were collected from broilers and tested by RRT-PCR and the HA gene were sequenced for the genetic evolution. Then this isolated virus was experimentally studied in a total of 20 SPF chicks (21 days old) that were divided into two equal groups. Group-1(G1) was negative control and group-2 (G2) was experimentally infected with the isolated LPAIV (H9N2). The obtained results revealed that all isolated LPAIV (H9N2) in this study were belong to G1-like Eurasian sub-lineages (A/quail/Hong Kong/G1/97-like) and clustered with Israeli strains in group B with specific mutation specific for Egyptian AIV-H9N2 isolated in 2018-2019 forming new subgroup-I. The experimental infection with LPAIV (H9N2) (G2) in SPF chickens, induced mild clinical signs, moderate histopathological changes in lung and spleen, shedding of higher mean LPAIV (H9N2) titre in trachea, up regulation of both the pro-inflammatory (IL-6) and the anti-viral (IFN-γ) cytokines, significant decrease of the serum levels of lysozyme and disturbance of the oxidative state where the serum nitric oxide (NO) and the malondialdehyde (MDA) levels were significantly increased and the antioxidants glutathione (GSH) and superoxide dismutase (SOD) were significantly decreased. In conclusion, the LPAIV (H9N2) was continually circulated in broiler chicken farms and highly evolved. The induced oxidative stress in LPAIV (H9N2) infection could be contributed to the viral pathogenesis, up-regulation of pro-inflammatory and anti-viral cytokines and down-regulation of the innate immune response.

Characterization of commercial poultry farms in Mexico: Towards a better understanding of biosecurity practices and antibiotic usage patterns.

Mexico is one of the world's major poultry producing countries. Two significant challenges currently facing the poultry industry are the responsible and judicious use of antimicrobials, and the potential occurrence of infectious disease outbreaks. For example, repeated outbreaks of highly pathogenic avian influenza virus subtype H7N3 have occurred in poultry since its first detection in Mexico in 2012. Both of these challenges can be addressed through good husbandry practices and the application of on-farm biosecurity measures. The aims of this study were: (i) to assess the biosecurity measures practiced across different types of poultry farms in Mexico, and (ii) to collect information regarding antimicrobial usage. A cross-sectional study was carried out through on-farm interviews on 43 poultry farms. A multiple correspondence analysis was performed to characterize the farms based on their pattern of biosecurity practices and antimicrobial usage. Three clusters of farms were identified using an agglomerative hierarchical cluster analysis. In each cluster, a specific farm type was predominant. The biosecurity measures that significantly differentiated the visited farms, thus allowing their clusterization, were: the use of personal protective equipment (e.g. face masks, hair caps, and eye protection), the requirement for a hygiene protocol before and after entering the farm, the use of exclusive working clothes by staff and visitors, footbath presence at the barn entrance, and the mortality disposal strategy. The more stringent the biosecurity measures on farms within a cluster, the fewer the farms that used antimicrobials. Farms with more biosecurity breaches used antimicrobials considered critically important for public health. These findings could be helpful to understand how to guide strategies to reinforce compliance with biosecurity practices identified as critical according to the farm type. We conclude by providing certain recommendations to improve on-farm biosecurity measures.

Development of ssDNA Aptamers for Diagnosis and Inhibition of the Highly Pathogenic Avian Influenza Virus Subtype H5N1

Avian influenza (AI) has severely affected the poultry industry worldwide and has caused the deaths of millions of birds. Highly pathogenic avian influenza virus is characterized by high mortality and the ability to transmit from birds to humans. Early diagnosis is difficult because of the variation in pathogenicity and the genetic diversity between virus subtypes. Therefore, development of a sensitive and accurate diagnostic system is an urgent priority. We developed ssDNA aptamer probes to detect AI viruses. Through seven rounds of SELEX to search for a probe specific to the highly pathogenic AI virus subtype H5N1, we identified 16 binding aptamers and selected two with the highest binding frequency. These two aptamers had strong binding affinities and low detection limits. We found that they could bind more specifically to H5N1, as compared to other subtypes. Furthermore, these aptamers inhibited hemagglutination, which is caused by the virus surface protein hemagglutinin. Our results indicate that our screened aptamers are effective molecular probes for diagnosing H5N1 and can be used as therapeutic agents to inhibit viral surface proteins. Sensitive diagnosis and suppression of avian influenza will help maintain a stable and healthy livestock industry, as well as protect human health.

Immunization of turkeys with a DNA vaccine expressing the haemagglutinin gene of low pathogenic avian influenza virus subtype H9N2

Low pathogenic avian influenza H9N2 is still circulating in the Middle East causing respiratory manifestations and severe economic losses in poultry. In the present study, an H9 plasmid-based DNA vaccine targeting the HA gene of H9N2 A/CK/Egypt/SCU8/2014 was developed and evaluated in turkeys. The full length of HA was cloned into vector plasmids under the control of a cytomegalovirus promoter. The in-vitro expression of the recombinant HA was demonstrated in HeLa cells transfected with the plasmids pVAX1-H9 or pCR-H9 using western blot and Immunofluorescent assay (IFA). The efficacy of pVAX-H9 and pCR- H9, naked or saponin-adjuvanted, was evaluated in turkey poults at 3 weeks and challenged with A/CK/Egypt/SCU8/2014 (106 EID50/bird at 3 weeks post-vaccination. The efficacy was assesses based on virus shedding, oropharyngeal and cloacal, as well as seroconversion using haemagglutination inhibition (HI) test. All immunized birds showed high HI antibody titers (7–8 log2) at 3 weeks post-vaccination. None of the birds vaccinated with naked or saponin-adjuvanted pVAX-H9 or pCR-H9 showed any clinical signs. The pVAX-H9 and pCR-H9 alone did not prevent cloacal and oropharyngeal virus shedding, however, saponin-adjuvanted pVAX1-H9 and pCR-H9 prevented cloacal and oropharyngeal virus shedding at 3 and 5 days post challenge, respectively. In conclusion, DNA vaccination with pVAX1-H9 and pCR-H9 could protect turkey from the H9N2 virus, but vaccination regimes need to be improved.