Avian Influenza (Bird Flu) Testing – Buyers Guide

Key Points:

  • Avian influenza is a notifiable disease listed by the OIE
  • There are 7 influenza genera but only influenza A viruses are known to infect birds 
  • Isolation of the virus in embryos has largely been replaced by validated molecular detection methods such as RT-PCR 
  • The type of swabs used may affect test sensitivity or validity with thin wire or plastic shafted swabs preferred
  • If using rapid tests make sure they have been validated for use on the type of bird to be tested 

Introduction: 
Avian Influenza virus (AIV) is a highly contagious virus in poultry and is the etiological agent of Avian Influenza (AI). The severity of the disease and its impact on poultry is dependent on the strain/subtype involved. H5 and H7 subtypes are the most common and they are categorized as Highly Pathogenic (HPAI) or Low-Pathogenic AI (LPAI) after its HA0 cleavage site amino acid sequence is determined. Symptoms of HPAI, for example, include blue discoloration of wattle and comb with depression, and common LPAI symptoms would be a drop in egg production also with depression. LPAIs have the potential to mutate into HPAIs.

Influenza in birds is caused by infection with viruses of the family Orthomyxoviridae placed in the genus Alphainfluenzavirus (influenzavirus A or influenza A virus) (International Committee on Taxonomy of Viruses (ICTV) 2019). Influenza A viruses are the only orthomyxoviruses known to naturally affect birds

It was first discovered in 1878 by an Italian researcher (Perroncito) as the cause of 'fowl plague.' The disease was known by this name until the international virology community changed its name to 'Avian influenza' at the first-ever symposium on the disease. Many AIV outbreaks over the last few centuries have resulted in the culling of great numbers of birds.

If the virus is not detected in time, it can make its way into the food chain and contaminate poultry products. There have been several incidents where the virus has crossed the species barrier and infected humans. In 1997, the HPAI H5N1 caused serious disease in Hong Kong in both humans and poultry. It resulted in 6 human fatalities and a total depopulation of poultry markets and chicken farms in the area. The transmission of the virus to humans is seldom from the consumption of meat but more from the meat processing environment. This buyer's guide will focus on AIV testing for poultry only.

Wild birds are AI’s natural reservoir and shed the virus via respiratory secretions and feces. Its path into the food chain comes from various factors, i.e., wild birds' contact with resident birds or through the importation of live poultry. The winter climate provides optimal conditions for the virus to proliferate in birds, and also facilitate its spread, as some birds migrate during this period from cold weather to milder temperatures.

Biosecurity measures such as feeding and watering birds under cover away from wild birds, changing bags used to collect litter, and monitoring birds for symptoms with testing are essential to prevent its spread and save bird lives. The LPAI subtype can become pathogenic if the virus pervades within the flock community for a long period of time. Veterinary vaccines for AI are available but limited. 

Testing for Avian Influenza virus:
Classic virus isolation: AI viral isolation in cell culture or embryonated eggs is the gold-standard technique because it is the most sensitive and accurate method, detecting only viable material and generating high titers of all types of AI viruses except the H17N10 virus. However, this method is time-consuming and not suitable for fast detection and routine diagnosis. Faster tests include RT-PCR, serological, laboratory-based antigen tests, and rapid near-patient/field antigen tests. 

Serological: Antibodies (Abs) to the AIV's glycoproteins: Hemagglutinin (HA), and Neuraminidase (NA), and also the nucleoprotein and matrix protein are produced in poultry after infection. These Abs can be detected by using immuno-diagnostic techniques:

  • Hemagglutination inhibition (HI) assay,
  • Neuraminidase inhibition (NI) assay,
  • Enzyme-linked immunoassay (ELISA),
  • Agar-Gel Immunodiffusion Assay (AGID)
  • Complement fixation (CF)
  • Neutralization test.

The HI and NI tests are inexpensive and commonly used for subtype identification and measuring the levels of respective antibodies to HA and NA in serum samples. ELISA tests are also commonly used to detect AI antibodies. Commercial ELISA kits are available and have been widely used for general serological monitoring of vaccinal responses. The AGID test can detect IgMs and IgGs. It is a low-cost serological screening test, but a high dose of antigens is required, and results can be observed after approximately 24–48 h. 

Lab-based Antigen Tests: These include ELISA and direct immunofluorescence on smears. However, these are in no way as sensitive as the cell culture method or RT-PCR. 

RT-PCR/NAT/Molecular tests: These kits require specimens isolated from nasal swabs, blood, urine, respiratory swabs, lung tissue, fecal samples, blood serum, tissue, or any body fluid. Specific primers and probes are available for each subtype, i.e., H5N1. Poultry farmers and veterinary labs would be notified of the subtypes in circulation by the World Health Organization (WHO), World Organisation for Animal Health (OIE), and genetic databases such as GeneBank and the Influenza Sequence database. 

Field/Point-of-Care Rapid Antigen Tests: There are many rapid antigen test kits on the market that detect over 15 subtypes of Influenza Type A virus in the tracheal and cloacal samples from symptomatic birds or flocks. The majority of these kits are lateral flow and use two antibodies specific to the nucleoprotein of Influenza Type A Virus. The anti-Influenza A antibody embedded in the test will bind to the Influenza A antigen present in the sample to form an antigen-antibody complex that migrates along a strip and is captured on a sensitized reaction line by the second antibody. This complex forms a visible line for a positive result. Despite the advantage of obtaining a result in 15 minutes, these rapid tests lack sensitivity or perhaps haven't been validated for different types of birds. OIE recommends these tests for field screening of high-mortality cases where AIV is suspected, but that a more sensitive lab-based test should be used for confirmation. 

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