Comparative Immunology, Microbiology and Infectious Diseases
Conventional and future diagnostics for avian influenza
Introduction
Avian influenza detection and diagnosis is comprised of a rapidly evolving combination of conventional methodologies and emerging technologies. The continued global spread of Asian H5N1 influenza virus since 2003, paired with the increasing awareness of pandemic potential, has resulted in commitment of significant resources toward improving and enhancing avian influenza (AI) virus detection tools. A choice of diagnostic technology can now be based on a combination of factors that includes fitness-for-purpose, technical ease, speed, diagnostic sensitivity, specificity, and cost. Tests for the specific diagnosis of AI virus can be divided into direct demonstration of the virus or indirect demonstration of virus exposure by detection of the specific antibody. Direct detection includes conventional culture of infectious virus, as well as the use of more rapid and cost-effective technologies that allow detection of specific viral antigens or nucleic acids. Advances in miniaturization, instrumentation, and computer analysis capabilities provide laboratories the ability to not only rapidly sub-type AI viruses, but also to rapidly perform complete genotypic and phylogenetic analysis on individual AI virus isolates. Antibody-based detection of AI virus exposure includes conventional agar-gel immunodiffusion and hemagglutination inhibition methods that have changed little since introduced decades ago, as well as advances in the well-established ELISA-based methodologies that provide on-site screening capabilities and potential for differential detection of avian influenza subtypes.
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Clinical diagnosis
Avian influenza infection can be complicated because the virus does not cause pathognomonic lesions either grossly or microscopically. To further complicate clinical diagnosis, asymptomatic infections are especially common in waterfowl species which serve as reservoirs of the virus. Asymptomatic carriers can however shed virus that is capable of causing severe lesions in other species, in particular domestic poultry. In poultry, pathological lesions are dependent on virus subtype, virus
Virus isolation and identification
Demonstration of infectious virus by inoculation of embryonated eggs is the historic gold standard for diagnosis of avian influenza virus, and generally follows internationally recognized methods [1]. The virus is inoculated into the chorioallantoic sac and in some cases additionally into yolk sac and onto chorioallantoic membrane [14] then incubated for periods of time ranging from 24 to 48 h for HPAI virus isolates, and up to 21 days through two or three blind passages for some LPAI virus
Serologic assays
Direct detection methods using molecular techniques or antigen capture have rapidly improved and become increasingly available in the same time that relatively few changes have been made in antibody-based detection approaches. The conventional serologic techniques of agar-gel immunodiffusion and hemagglutination inhibition continue to be widely used globally for surveillance and disease control efforts in domestic poultry species [1]. A competitive ELISA (cELISA) has been described as a
Environmental sampling
For AI virus detection and diagnostic methods, specimen collection occurs at the individual bird level, though for cost efficiency and interpretation of results samples may be pooled, statistical sampling approaches applied, and the flock considered as the diagnostic unit of interest. An alternate approach for sampling to detect avian influenza virus has been proposed. Environmental air-sampling paired with realtime RT-PCR was used to detect exotic Newcastle disease virus in commercial poultry
Conclusions
Avian influenza virus is recognized as a highly mutable virus that is constantly evolving. Although the conventional methods of virus isolation, identification, and pathotyping have stood the test of time, new technologies and approaches for AI virus diagnosis also continue to evolve. Antigen detection and molecular techniques continue to speed the ability to not only detect AI virus from clinical specimens, but to provide in depth phylogenetic and pathotype information in minutes to hours. The
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