Abstract Avian Influenza Research Articles

High pathogenicity avian influenza: targeted active surveillance of wild birds to enable early detection of emerging disease threats.

Avian influenza (AI) is an important disease that has significant implications for animal and human health. High pathogenicity AI (HPAI) has emerged in consecutive seasons within the UK to cause the largest outbreaks recorded. Statutory measures to control outbreaks of AI virus (AIV) at poultry farms involve disposal of all birds on infected premises. Understanding of the timing of incursions into the UK could facilitate decisions on improved responses. During the autumnal migration and wintering period (autumn 2019- spring 2020), three active sampling approaches were trialled for wild bird species considered likely to be involved in captive AI outbreaks with retrospective laboratory testing undertaken to define the presence of AIV.Faecal sampling of birds (n = 594) caught during routine and responsive mist net sampling failed to detect AIV. Cloacal sampling of hunter-harvested waterfowl (n = 146) detected seven positive samples from three species with the earliest detection on the 17 October 2020. Statutory sampling first detected AIV in wild and captive birds on 3 November 2020. We conclude that hunter sourced sampling of waterfowl presents an opportunity to detect AI within the UK in advance of outbreaks on poultry farms and allow for early intervention measures to protect the national poultry flock.

Infectivity and pathobiology of H7N1 and H5N8 high pathogenicity avian influenza viruses for pigeons (Columba livia var. domestica)

ABSTRACT Avian influenza (AI) is one of the most important viral diseases in poultry, wildlife and humans. Available data indicate that pigeons play a minimum role in the epidemiology of AI. However, a degree of variation exists in the susceptibility of pigeons to highly pathogenic AI viruses (HPAIVs), especially since the emergence of the goose/Guangdong H5 lineage. Here, the pathogenesis of H5N8 HPAIV in comparison with a H7N1 HPAIV and the role of pigeons in the epidemiology of these viruses were evaluated. Local and urban pigeons (Columba livia var. domestica) were intranasally inoculated with 105 ELD50 of A/goose/Spain/IA17CR02699/2017 (H5N8) or A/Chicken/Italy/5093/1999 (H7N1) and monitored during 14 days. Several pigeons inoculated with H5N8 or H7N1 seroconverted. However, clinical signs, mortality, microscopic lesions and viral antigen were only detected in a local pigeon inoculated with H5N8 HPAIV. This pigeon presented prostration and neurological signs that correlated with the presence of large areas of necrosis and widespread AIV antigen in the central nervous system, indicating that the fatal outcome was associated with neurological dysfunction. Viral RNA in swabs was detected in some pigeons inoculated with H7N1 and H5N8, but it was inconsistent, short-term and at low titres. The present study demonstrates that the majority of pigeons were resistant to H5N8 and H7N1 HPAIVs, despite several pigeons developing asymptomatic infections. The limited viral shedding indicates a minimum role of pigeons as amplifiers of HPAIVs, regardless of the viral lineage, and suggests that this species may represent a low risk for environmental contamination. RESEARCH HIGHLIGHTS H7N1 and H5N8 HPAIVs can produce subclinical infections in pigeons. The mortality caused by H5N8 HPAIV in one pigeon was associated with neurological dysfunction. Pigeons represent a low risk for environmental contamination by HPAIVs.

Broilers of live bird markets at Mymensingh city carrying H9N2 Avian influenza virus in their respiratory tract

Avian influenza (AI) caused by Type A influenza virus is a global zoonosis, infecting vast majority of mammalian and avian species. Broilers are meat type birds and randomly reared and sold by the farmers in Bangladesh with poor biosecurity. This study was aimed to identify the Type and subtypes of AI viruses in the broilers of two live bird markets, Mymensingh. A total of 10 birds from each of the market were randomly selected, investigated by clinical, pathological, reverse transcriptase polymerase chain reactions (RT-PCR), sequencing and sequence analysis. Out of 20 birds investigated, 06 were sick, 02 were dead and 12 were apparently healthy. Clinically, the sick/dead birds did not reveal any changes typical to AI. During necropsy, the sick/dead birds showed congested lungs and moderate hemorrhages in the trachea. Such lesions was absent in the lungs of apparently healthy birds. Following histopathological examination interstitial pneumonia with bronchitis was seen in sick/dead birds. The RT-PCR protocol was adapted to identify matrix protein gene of Type A influenza virus and amplified 430bp fragment is even cases. To identify the sub types of AI viruses involved, hemagglutinin (HA) and neuraminidase (NA) gene specific RT-PCR was carried out. 1475bp and 1089bp amplicons specific to HA and NA genes of AI viruses were generated in 07 cases. The cDNAs of HA and NA genes were sequenced, edited and revealed that the AI virus circulated in the live bird market of Mymensingh city is H9N2 subtype. Two sick, one dead and four apparently healthy birds found to carry H9N2 AI virus. The H9N2 virus is naturally low pathogenic for poultry, has got public health significance, and may donate partial or even whole cassette of internal genes to generate novel human-lethal reassortants of AI viruses; this was main concern for AI viral outbreak investigation in this study. It needs to examine large number of samples from wider sources to trace the rate of mutation and subsequent reemergence of pandemic AI viruses.Res. Agric., Livest. Fish.5(2): 225-233, August 2018

Effectiveness of different avian influenza (H5) vaccination regimens in layer chickens on the humoral immune response and interferon-alpha signalling immune marker.

Avian influenza (AI) vaccines are widely used to control and eliminate the ongoing avian influenza virus epidemic in Egypt. A strict vaccination policy with inactivated AI vaccines has been widely applied, however the virus still circulating, evolving and causing great negative impact to the poultry sector in Egypt. Therefore, an updated poultry vaccination policy using different vaccine technologies might be valuable as an innovative additional control strategy of AIV in Egypt. In the present study, the effectiveness of different avian influenza (AI) vaccination schedules was evaluated in 300 commercial layer chicks (ISA White) using either the oil-emulsion baculovirus-H5-prototype vaccine (baculovirus-H5 prototype) or turkey herpesvirus (HVT) vector vaccine containing the hemagglutinin (HA) gene from H5N1 strain (rHVT-H5), applied alone or in combination and in different settings. Vaccination with either two injections of the baculovirus-H5 prototype, a single injection of rHVT-H5 or priming with rHVT-H5 at 1day old followed by boosting with the baculovirus-H5 prototype induced AI-HI protective antibody responses starting as early as 3 to 4weeks of age and lasting up to the end of the rearing period (16weeks). A single vaccination with the baculovirus-H5 prototype did not generate a protective antibody titre for the entire rearing period. Furthermore, the present study elucidated that vaccination once or twice with the baculovirus-H5 vaccine prototype activated the chicken interferon-alpha (Ch-IFN-alpha) signalling pathway via transduction of antiviral components, e.g., Mx1 and IRF7. Birds immunized once with rHVT-H5 at 1day old did not show activation of the Mx1 and IRF7 transcripts; however, following boosting with the baculovirus-H5 prototype vaccine, up-regulation of Mx1 and IRF7 was observed. Based on our findings, it can be concluded that either reinforcement with two injections of the baculovirus-H5 prototype or prime-boost vaccination (rHVT-H5 at 1day old followed by the baculovirus-H5 prototype vaccine at 8days old) is a successful strategy to induce both innate and humoral immune responses and could be recommended for the layer production sector over the entire rearing period, especially in AI-endemic areas.

Positive regulation of humoral and innate immune responses induced by inactivated Avian Influenza Virus vaccine in broiler chickens.

Avian Influenza (AI) vaccines are widely used for mammals and birds in a trial to eliminate the Avian Influenza virus (AIV) infection from the world. However and up till now the virus is still existed via modulation of its antigenic structure to evade the pressure of host immune responses. For a complete understanding of the immune responses following AI vaccination in chickens, the modulations of the chickens humoral immune responses and interferon-alpha signaling pathway, as a fundamental part of the innate immune responses, were investigated. In our study, we measured the humoral immune response using hemagglutination-inhibition (HI) and enzyme-linked immunosorbent assay (ELISA) tests. In addition, chicken interferon-alpha pathway components was measured at RNA levels using Quantitative Real-time PCR (qRT-PCR) following one dose of inactivated H5N1 influenza vaccine at 14 days of age. In this study, the protective levels of humoral antibody responses were observed at 14, 21 and 28 days following immunization with inactivated (Re-1/H5N1) AI vaccine. In the chicken spleen cells, up regulation in the chicken interferon-alpha pathway components (MX1 & IRF7) was existed as early as 48 h post vaccination and remained until 28 days post vaccination at the endogenous state. However, after the recall with ex-vivo stimulation, the up regulation was more pronounced in the transcriptional factor (IRF7) compared to the antiviral gene (MX1) at 28 days post vaccination. So far, from our results it appears that the inactivated H5N1 vaccine can trigger the chicken interferon-alpha signaling pathway as well as it can elicit protective humoral antibody responses.

AVIAN INFLUENZA AND ITS MASS DEPOPULATION STRATEGIES IN INFECTED POULTRY BIRDS

Avian influenza (AI) is a highly contagious and zoonotic viral disease that affects several animal species. It causes heavy economic losses in the domestic poultry. A quick response is always desired in the event of any disease outbreak. The principal approach to control a contagious disease involves the killing of diseased animals along with the bio containment of infectious agent. Mass depopulation of the infected birds plays an important role in the eradication of the disease. The possible strategies for mass depopulation include maceration, electrocution, cervical dislocation, gassing and foaming. All of these procedures are much intensive and time consuming because it involves a lot of man power, biosecurity risks, applicability for all house types and suitability for large-scale emergency implementation. The basic objectives of these strategies include (1) To reduce pain and suffering to the birds, (2) To minimize disease spread and (3) To ensure of protection to human operators from potential biohazards. A suitable depopulation technique can only be suggested keeping in view the species and type of bird involved, and differences in husbandry practices like management, housing and stocking density. Mass depopulation is an important tool to control the spread of any disease but the selection of procedure depends upon the prevailing circumstances. In this paper, various mass depopulation strategies and their selection in different conditions is reviewed and discussed.

Abiotic Factors Affecting the Persistence of Avian Influenza Virus in Surface Waters of Waterfowl Habitats

Avian influenza (AI) virus can remain infectious in water for months, and virus-contaminated surface water is considered to be a source of infection within wild waterfowl populations. Previous work has characterized the effects of pH, salinity, and temperature on viral persistence in water, but most of that work was done with modified distilled water. The objective of this study was to identify the abiotic factors that influence the duration of AI virus persistence in natural surface water. Surface water samples were collected from 38 waterfowl habitats distributed across the United States. Samples were submitted to the U.S. Geological Survey National Water Quality Laboratory for chemical analysis and the University of Georgia for viral reduction time analysis. Samples were filtered with 0.22-μm filters, and the durations of persistence of three wild-bird-derived influenza A viruses within each water sample at 10, 17, and 28°C were determined. The effects of the surface water physicochemical factors on the duration of AI viral persistence in laboratory experiments were evaluated by multivariable linear regression with robust standard errors. The duration of AI virus persistence was determined to be longest in filtered surface water with a low temperature (<17°C), a neutral-to-basic pH (7.0 to 8.5), low salinity (<0.5 ppt), and a low ammonia concentration (<0.5 mg/liter). Our results also highlighted potential strain-related variation in the stability of AI virus in surface water. These results bring us closer to being able to predict the duration of AI virus persistence in surface water of waterfowl habitats.

Emergence of a Highly Pathogenic Avian Influenza Virus from a Low-Pathogenic Progenitor

Avian influenza (AI) viruses of the H7 subtype have the potential to evolve into highly pathogenic (HP) viruses that represent a major economic problem for the poultry industry and a threat to global health. However, the emergence of HPAI viruses from low-pathogenic (LPAI) progenitor viruses currently is poorly understood. To investigate the origin and evolution of one of the most important avian influenza epidemics described in Europe, we investigated the evolutionary and spatial dynamics of the entire genome of 109 H7N1 (46 LPAI and 63 HPAI) viruses collected during Italian H7N1 outbreaks between March 1999 and February 2001. Phylogenetic analysis revealed that the LPAI and HPAI epidemics shared a single ancestor, that the HPAI strains evolved from the LPAI viruses in the absence of reassortment, and that there was a parallel emergence of mutations among HPAI and later LPAI lineages. Notably, an ultradeep-sequencing analysis demonstrated that some of the amino acid changes characterizing the HPAI virus cluster were already present with low frequency within several individual viral populations from the beginning of the LPAI H7N1 epidemic. A Bayesian phylogeographic analysis revealed stronger spatial structure during the LPAI outbreak, reflecting the more rapid spread of the virus following the emergence of HPAI. The data generated in this study provide the most complete evolutionary and phylogeographic analysis of epidemiologically intertwined high- and low-pathogenicity viruses undertaken to date and highlight the importance of implementing prompt eradication measures against LPAI to prevent the appearance of viruses with fitness advantages and unpredictable pathogenic properties. The Italian H7 AI epidemic of 1999 to 2001 was one of the most important AI outbreaks described in Europe. H7 viruses have the ability to evolve into HP forms from LP precursors, although the mechanisms underlying this evolutionary transition are only poorly understood. We combined epidemiological information, whole-genome sequence data, and ultradeep sequencing approaches to provide the most complete characterization of the evolution of HPAI from LPAI viruses undertaken to date. Our analysis revealed that the LPAI viruses were the direct ancestors of the HPAI strains and identified low-frequency minority variants with HPAI mutations that were present in the LPAI samples. Spatial analysis provided key information for the design of effective control strategies for AI at both local and global scales. Overall, this work highlights the importance of implementing rapid eradication measures to prevent the emergence of novel influenza viruses with severe pathogenic properties.

Comparison of single 1-day-old chick vaccination using a Newcastle disease virus vector with a prime/boost vaccination scheme against a highly pathogenic avian influenza H5N1 challenge

Avian influenza (AI) vaccines should be used as part of a whole comprehensive AI control programme. Vectored vaccines based on Newcastle disease virus (NDV) are very promising, but are so far licensed in only a few countries. In the present study, the immunogenicity and protection against a highly pathogenic H5N1 influenza challenge were evaluated after vaccination with an enterotropic NDV vector expressing an H5 haemagglutinin (rNDV-H5) in 1-day-old specific pathogen free chickens inoculated once, twice or once followed by a heterologous boost with an inactivated H5N9 vaccine (iH5N9). The heterologous prime/boost rNDV-H5/iH5N9 combination afforded the best level of protection against the H5N1 challenge performed at 6 weeks of age. Two rNDV-H5 administrations conferred a good level of protection after challenge, although only a cellular H5-specific response could be detected. Interestingly, a single administration of rNDV-H5 gave the same level of protection as the double administration but without any detectable H5-specific immune response. In contrast to AI immunity, a high humoral, mucosal and cellular NDV-specific immunity could be detected up to 6 weeks post vaccination after using the three different vaccination schedules. NDV-specific mucosal and cellular immune responses were slightly higher after double rNDV-H5 vaccination when compared with single inoculation. Finally, the heterologous prime/boost rNDV-H5/iH5N9 combination induced a broader detectable immunity including systemic, mucosal and cellular AI and NDV-specific responses.

Development of H5 subtype-specific monoclonal antibodies (MAb) and MAb-based assays for rapid detection of H5 avian influenza

Avian influenza (AI) virology surveillance is the most important method to monitor AI virus (AIV) in poultry so as to effectively prevent and control AI outbreaks. Monoclonal antibodies (MAb)-based assays are highly sensitive and specific for AIV detection, and much practical and economic for test-in-field or onsite. Many such assays have been developed and are still in developing since the H5N1 highly pathogenic AI (HPAI) outbreaks occurred in South East Asia in 2003. A MAb-based dot-enzyme-linked immunosorbent assay (ELISA) has been developed in our lab during late 1990s and early 2000s. Meanwhile, AIV H7 and H5 subtype specific-MAbs have been successfully developed in our laboratory to enhance the Dot-ELISA and other MAb-based assays for AIV detection. Production and purification of the H7 and H5 MAbs were made to provide essential reagents for Dot-ELISA and other immunoassays, and the current development of a novel Biosensor technique for rapid detection of AIV from clinical and field specimens.

Molecular characterization and phylogenetic analysis of neuraminidase gene of avian Influenza H9N2 viruses isolated from commercial broiler chicken in Iran during a period of 1998-2007

Avian influenza (AI) outbreaks due to H9N2 subtype of avian influenza virus (AIV) occurred in poultry industry in Iran, throughout the past decade and caused serious economic losses as a result of decreased egg production, increased mortality and the cost of vaccination in Iranian poultry industry. Our aim was to find the phylogenic and genetic relationships among avian influenza viruses recently isolated from poultry in Iran with other Asian H9N2 lineages (1998-2007). The complete nucleotide sequences of the neuraminidase (NA) genes of ten H9N2 influenza virus strain isolated from diseased chickens in different farms in Iran during 1998-2007, were amplified and sequenced and phylogenetic relationships between these and previously reported N2 genes were investigated. The viruses surface glycoprotein genes was >90% similar to those of A/Quail/Hongkong/G1/97 (H9N2) lineage. The NA stalks regions in these viruses had no deletion or insertion or shortening as compared to that A/DK/HK/Y280/97 lineage (CK/Bei-like viruses) and the 2 human isolates A/HK/1073/99, A/HK/1074/99. The hemadsorbing site of NA had up to 3 amino acid substitution and is different from those of earlier Iranian viruses and ten Iranian isolates possessed seven potential glycosylation sites like the most H9N2 isolates. Phylogenetic analysis of NA gene showed that they belonged to the same A/Quail/Hong Kong/G1/97 like virus sub lineage. Key words: Avian influenza virus, H9N2, neuraminidase gene, phylogenetic analysis, Iran.

Proteomic Analysis of the Serum from Chicken Infected by Avian Influenza Virus

Avian influenza (AI) is an infectious, low pathogenic virus that is endemic all over the world and poses a potential threat to the poultry industry. Vaccination is a widely used effective method to prevent avian influenza virus. Here we employed a comparative proteomics approach [two-dimensional electrophoresis (2-DE) and matrix assisted laser desorption ionization-time of flight (MALDI-TOF)] to characterize proteome in the sera from the specific pathogen free (SPF) chickens, the vaccinated chickens, and the naturally infected chickens. We identified total 58 proteins that were differentially expressed in the sera of three groups. Among them ovotransferrin and vitamin D-binding protein were more expressed in the sera of naturally infected chickens compare with other groups. Our results suggested that the level of these two proteins in the serum may help to discriminate the naturally infected chicken from the vaccinated chicken.

Detection of the Avian Influenza Viruses Nonstructural Protein 1 for Distinction between Vaccinated and Infected Chickens Using Synthetic Peptide-Based ELISA

Avian influenza (AI) virus infects both animal and human. Low pathogenic AI virus infections (some H7 and H9 subtypes) have been reported all over the world and pose a potential threat to the poultry industry. Vaccination is the most effective way to prevent virus infection. However, vaccination makes it difficult to differentiate between vaccinated chickens and infected chickens. In order to differentiate vaccinated chickens from naturally infected chickens, we adopted synthetic peptide-based enzyme-linked immunosorbent assay (ELISA) using the peptide sequences from nonstructural protein 1 (NS1) of H9N2. Five synthetic peptides were designed using Protein Variability Sever (http://imed.med.ucm.es/PVS/) and synthesized. NS1-1 ~ NS1-4 peptides failed to detect serum antibodies from both vaccinated and naturally infected chickens. NS1-5 peptide from the C-terminal NS1 protein detected serum antibody from naturally infected chickens but not vaccinated chickens. These results imply that NS1-5 peptide may be a useful tool to differentiate naturally infected chicken from vaccinated chicken as being used in the synthetic peptide-based ELISA.

Europeanization, Uploading and Downloading

Avian influenza (AI) has called for transnational policy making by member state governments and the EU as outbreaks of the disease have spread across state boundaries. The focus of this article is on the relationship between the UK and EU surrounding the AI case study from 2005 to early 2007. The analysis is informed by interviews conducted with officials based in the Department for the Environment, Food and Rural Affairs (Defra) who have been involved in projecting or `uploading' national policy preferences to the European Commission and, at the same time, accommodating or `downloading' the rules and regulations that have emanated mainly from the Standing Committee for Food Chain and Animal Health (SCFCAH) of the Commission. The article, therefore, adopts a dynamic view of Europeanization processes in order to analyse transnational policy making for AI outbreaks. The article shows that dynamic Europeanization processes are effective in conceptualizing and explaining transnational policy making for AI across the UK and EU levels.

Animal and Human Health Implications of Avian Influenza Infections

Avian influenza (AI) is a listed disease of the World Organisation for Animal Health (OIE) that has become a disease of great importance both for animal and human health. Until recent times, AI was considered a disease of birds with zoonotic implications of limited significance. The emergence and spread of the Asian lineage highly pathogenic AI (HPAI) H5N1 virus has dramatically changed this perspective; not only has it been responsible of the death or culling of millions of birds, but this virus has also been able to infect a variety of non-avian hosts including human beings. The implications of such a panzootic reflect themselves in animal health issues, notably in the reduction of a protein source for developing countries and in the management of the pandemic potential. Retrospective studies have shown that avian progenitors play an important role in the generation of pandemic viruses for humans, and therefore these infections in the avian reservoir should be subjected to control measures aiming at eradication of the Asian H5N1 virus from all sectors rather than just eliminating or reducing the impact of the disease in poultry.

Emergency Assistance in Laboratory Development for Control of Avian Influenza in Laos and Cambodia

Avian influenza (AI) diagnostic laboratories in Laos and Cambodia have been established recently to conduct AI surveillance and diagnostic tests to coordinate regional efforts for the control of highly pathogenic avian influenza (HPAI) in South East Asia. Two laboratories have been provisioned with equipment, supplies, and reagents for routine diagnostic testing. Laboratory staff has received training to conduct serologic and virologic tests for isolation and identification of AI virus. Development of a disease reporting system and an AI surveillance program is in progress in Laos and Cambodia. There are plans to further upgrade laboratory facilities and to provide more comprehensive and advanced molecular diagnostic tests for control of HPAI in Laos and Cambodia. These two countries are on the frontline in the battle to fight HPAI H5N1 virus and to prevent it from spreading to other regions and mutating to a major human pathogen.

Assessing the Probability of the Presence of Low Pathogenicity Avian Influenza Virus in Exported Chicken Meat

Avian influenza (AI) is a disease of concern for the poultry industry. In its highly pathogenic form, AI viruses (AIVs) can cause a high morbidity and case fatality rate as well as severe economic consequences. Low pathogenic AIVs (LPAIVs), in contrast, only cause localized infections in the respiratory and gastrointestinal tracts of affected birds. Although there is apparently sufficient scientific evidence documenting the absence of LPAIV in poultry meat, several countries still place restrictions for international trade of poultry meat on LPAIV-infected countries. These restrictions are extremely trade disruptive and entail significant losses to the poultry industry. This article presents a quantitative approach to assess the probability of LPAIV presence in chicken meat and provides a model that can be tailored to reflect the epidemiology of LPAIV and surveillance systems in different countries. Results show that the probability of introducing LPAIV through chicken meat imports is insignificant.

Detection of Avian Influenza Virus of H9 Subtype in the Faeces of Experimentally and Naturally Infected Chickens by Reverse Transcription-Polymerase Chain Reaction

Avian Influenza (AI) is a viral, highly contagious disease of domestic and wild birds. In an avian diagnostic laboratory, it is essential to have methods for rapid detection of respiratory viruses. In the present study, cloacal swabs collected from chickens experimentally and naturally infected with mild pathogenicity AI virus subtype H9, used in a reverse transcription-polymerase chain reaction (RTPCR) assay for detection of AI. On cloacal swabs collected from experimentally infected chickens, AI virus was detected most frequently between days 3 and 7 post infection (p.i.) and the relative sensitivity, specificity, correlation rate, positive predictive value and negative predictive value of the RT-PCR compared to virus isolation (VI) assay were 84%, 80%, 82%, 83% and 81%, respectively. On pooled cloacal swabs collected from flocks suspected of AI, these results were 96%, 100%, 97%, 83% and 100%, respectively. The results proved that the RT-PCR assay could be a reliable and rapid alternative to VI assay for detection of AI virus subtype H9 in faecal specimens.

Development and validation of an anti-N3 indirect immunofluorescent antibody test to be used as a companion diagnostic test in the framework of a “DIVA” vaccination strategy for avian influenza infections in poultry

Avian influenza (AI) infections have become of growing importance both for animal and human health. Vaccination has become a recommended tool to support eradication efforts and limit the economic losses caused by this disease. The "DIVA" system, using a vaccine containing a heterologous neuraminidase to the field virus, has been shown to be an effective tool in increasing the resistance of birds to field challenge, preventing clinical signs and reducing viral shedding in the environment. The companion diagnostic test to the vaccine, however, has been only partially validated in the field against one subtype of neuraminidase (N1). The present paper presents the results of a full laboratory and field validation of the diagnostic test developed to detect antibodies to the N3 subtype of AI in vaccinated and unvaccinated chickens and turkeys. Antibody kinetic studies conducted in the laboratory have shown that antibodies to the N protein may be detected earlier than antibodies to the haemagglutinin. The data derived from this extensive validation trial indicate the excellent capability of this assay in detecting the presence of active AI infection at an early stage in both unvaccinated and vaccinated birds and the lack of interference with vaccine-induced antibodies.

Re-Evaluation of the Pathogenicity of A/Chicken/Alabama/75 (H4N8) Influenza Virus

Avian influenza (AI) virus A/chicken/Alabama/7395/75 (H4N8), a putatively non-pathogenic virus associated with a self-limiting outbreak of severe disease in commercial layers, was selectively passed in chickens or in cell cultures and then in chickens to determine whether virus with increased pathogenicity would emerge. When 20 derivatives of the parental virus were each inoculated intranasally and intratracheally in leghorn hens, mortality rates ranged from zero (0/24) to 25% (6/24); mortality was 4% (1/24) for hens inoculated with the parental virus. Many virus reisolates (51/144) from hens that died exhibited high pathogenicity, killing at least six of eight intravenously inoculated 4-week-old chickens. Most derivatives examined produced plaques in trypsin-free cell cultures more efficiently than the parental virus, but the highest plaquing efficiencies observed (10%) were lower than would be expected (100%) for highly pathogenic subtype H5 or H7 AI viruses. These results confirm that the Alabama H4N8 virus can acquire increased pathogenicity upon passage in chickens and suggest that it may have acted alone in producing the severe disease observed in laying chickens in Alabama.