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Various metrics have been used to directly measure vaccine-induced protection, including prevention of death, clinical signs, and lesions; prevention of decreases in egg production and alterations in egg quality; quantification of the reduction in virus replication and shedding from the respiratory tract and gastrointestinal tracts; and prevention of contact transmission in in vivo poultry experiments. In addition, indirect measures of vaccine potency and protection have been developed and validated against the direct measures and include serological assays in vaccinated poultry and the assessment of the content of HA antigen in the vaccine. These indirect assessments of protection are useful in determining if vaccine batches have a consistent ability to protect. For adequate potency, vaccines should contain 50 mean protective doses of antigen per dose, which corresponds to 0.3-7.8 μg of HA protein in inactivated vaccines, depending on immunogenicity and antigenic relatedness of individual seed strains.Avian infection studies with influenza A are an important means of assessing host susceptibility, viral pathogenesis, host responses to infection, mechanisms of transmission, viral pathotype, and viral evolution. Complex systems and natural settings may also be explored with carefully designed infection studies. In this chapter, we explore the elements of infection studies, general guidelines for choosing a virus to use, host selection, and many aspects of study design.ELISA assays are a fast and relatively inexpensive way to screen sera for antibodies to avian influenza virus. Commercial ELISA kits are available, and although they are more expensive than in-house tests, they provide a ready-to-use assay with good quality control. Numerous sample types can be processed for ELISA serum, plasma, egg yolk, or blood collected on filter paper. High-quality samples are critical to accurate results. The basics of AIV antibody ELISA, sample processing, result interpretation, and troubleshooting are discussed.The agar gel immunodiffusion (AGID) test is used to detect antibodies to Type A influenza group-specific antigens, i.e., the nucleoprotein (NP) and matrix (M) proteins. Therefore, this test will detect antibodies to all influenza A virus subtypes. AGID is commonly used to screen poultry flocks for avian influenza virus infection. The AGID is a simple and economical serological test. All serological testing has its advantages and disadvantages, which should be considered before choosing the optimal test for the laboratory needs. Each laboratory must evaluate the laboratory's resources, the volume of testing, the goal of testing, how the test results are used, and what types of samples are being tested in order to select the optimal test.Avian influenza virus and some mammalian influenza A viruses can be isolated, propagated, and titrated in embryonated chicken eggs (ECEs). Most sample types can be accommodated in ECE culture with appropriate processing. Isolation may also be accomplished in cell culture, and if a mammalian lineage influenza A is expected to be in an avian sample, for example swine influenza in turkey specimens, mammalian cell may be preferable. Culture in ECEs is highly sensitive but is not specific for influenza A, which may be an advantage because a sample may be screened for several agents at once. Once an agent is isolated in culture, the presence of influenza viruses can be confirmed by hemagglutination inhibition assay, antigen immunoassay, agar gel immunodiffusion assay, or RT-PCR. Finally, ECEs may be used to propagate and titrate an avian influenza virus.Real-time RT-PCR (rRT-PCR) has been used for avian influenza virus (AIV) detection since the early 2000s. This method has been applied to surveillance, outbreaks and research. QVDOph Some of the advantages of rRT-PCR are high sensitivity, high specificity, rapid time to result, scalability, cost, and its inherently quantitative nature. Furthermore, rRT-PCR can be used with numerous sample types and is less expensive than virus isolation in chicken embryos, and since infectious virus is inactivated early during processing, biosafety and biosecurity are also easier to maintain. However, the high genetic variability of AIV may decrease sensitivity and increases the chances of a false negative result with novel strains. This chapter will provide an overview of the USDA-validated rRT-PCR procedure for the detection of type A influenza.The efficient extraction and purification of viral RNA is critical for downstream molecular applications such as the sensitive and specific detection of virus in clinical samples, virus gene cloning and expression, gene sequencing, or quantification of avian influenza (AI) virus by molecular methods from experimentally infected birds. Samples can generally be divided into two types enriched (e.g., virus stocks) and non-enriched (e.g., clinical). Clinical samples, which may be tissues or swab material, are the most difficult to process due to the complex sample composition and possibly low virus titers. In this chapter, two well-established procedures for the extraction of AI virus RNA from common clinical specimen types and enriched virus stocks will be presented.Successful detection of avian influenza (AI) virus, viral antigen, nucleic acid, or antibody is dependent upon the collection of the appropriate sample type, the quality of the sample, and the proper storage and handling of the sample. The diagnostic tests to be performed should be considered prior to sample collection. Sera are acceptable samples for ELISA or agar gel immunodiffusion tests, but not for real-time RT-PCR. Likewise, swabs and/or tissues are acceptable for real-time RT-PCR and virus isolation. The sample type will also depend on the type of birds that are being tested; oropharyngeal swabs from gallinaceous poultry and cloacal swabs from waterfowl are the preferred specimens for most diagnostic tests, although it is optimal to collect swabs from both locations, if possible. In addition to collecting the appropriate sample for the tests to be performed, selecting the right materials for sample collection (i.e., type of swab) is very important. This chapter will outline the collection of different specimen types and procedures for proper specimen handling.