Diagnostic testing plays a key role in a surveillance program as diagnostic testing aims to accurately determine the infection or disease status of an individual animal. Diagnostic assays for AIV can be categorized into four broad types: tests for detecting the virus, its antigen, its genomic material, and antibodies to the virus. Virus characterization almost always follows virus detection. The present article surveys the current literature on the goals, principles, test performance, advantages, and disadvantages of these diagnostic assays. Virus isolation can be achieved using embryonating eggs or cell cultures in a lab setting. Virus antigens can be detected by antigen-capturing immunoassays or tissue immunoassays. Viral RNA can be detected by PCR-based assays (gel-based reverse transcription–polymerase chain reaction (RT-PCR), or probe or SYBR^® Green-based real-time RT-PCR), loop-mediated isothermal amplification, in situ hybridization, and nucleic acid sequence-based amplification. Antibodies to AIV can be detected by ELISA, agar gel immunodiffusion, hemagglutination inhibition, and microneutralization. Avian influenza virus can be characterized by hemagglutination inhibition, neuraminidase inhibition, sequencing (dideoxynucleotide chain-termination sequencing, next-generation sequencing), genetic sequence-based pathotype prediction, and pathogenicity testing. Novel and variant AIVs can be recognized by DNA microarrays, electron microscopy, mass spectroscopy, and Biological Microelectromechanical Systems. A variety of diagnostic tests are employed in AIV surveillance and monitoring. The choice of their use depends on the goal of testing (fit for purpose), the time of testing during the disease, the assay target, the sample matrix, assay performance, and the advantages and disadvantages of the assay. The article concludes with authors’ perspective of the use of diagnostic assays in the surveillance and monitoring of AIV in poultry.

Reports of a mass die-off of ∼350 elephants (Loxodonta africana) in northern Botswana over a period of two months (May–June 2020), has fuelled speculation and concern regarding the cause. Although the area in which these mortalities occurred is not protected and is considered a hotspot for human–elephant conflict and poaching, both malicious poisoning and poaching are unlikely to have played a role as other species were not affected, and elephant carcasses were found with tusks intact. In the absence of a confirmed cause we sought to identify the lines of enquiry that are most likely to lead to a definitive answer. In particular, we consider viral and bacterial agents that could precipitate species-specific mortalities on this scale, potential environmental sources of poisoning and the samples and tests that would assist in excluding/ confirming these candidate causes. Whilst it may be argued that these mortalities are unlikely to negatively impact the broader elephant population of ∼130 000 individuals in Botswana, the same cannot be said of the many vulnerable population pockets in other parts of Africa. For this reason, it is essential that the cause of the current die-off is identified as it is the only way to prevent similar losses of susceptible elephants elsewhere.

The present study evaluated the potential utility of feather samples for the convenient and accurate detection of avian influenza virus (AIV) in commercial poultry. Feather samples were obtained from AIV-negative commercial layer facilities in Iowa, USA. The feathers were spiked with various concentrations (10⁶ to 10⁰) of a low pathogenic strain of H5N2 AIV using a nebulizing device and were evaluated for the detection of viral RNA using a real-time RT-PCR assay immediately or after incubation at −20, 4, 22, or 37 °C for 24, 48, or 72 h. Likewise, cell culture medium samples with and without the virus were prepared and used for comparison. In the spiked feathers, the PCR reliably (i.e., 100% probability of detection) detected AIV RNA in eluates from samples sprayed with 10³ EID₅₀/mL or more of the virus. Based on half-life estimates, the feathers performed better than the corresponding media samples (p < 0.05), particularly when the samples were stored at 22 or 37 °C. In conclusion, feather samples can be routinely collected from a poultry barn as a non-invasive alternative to blood or oropharyngeal–cloacal swab samples for monitoring AIV.