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April 2008 Issue

Avian Influenza: Signs, Transmission and Monitoring

Avian Influenza
Avian influenza (AI) is a disease of both wild and domestic poultry caused by viruses of the family Orthomyxoviridae, genus Influenzavirus A. AI viruses can infect chickens, turkeys, pheasants, quail, ducks, geese, guinea fowl and a variety of other birds, including migratory waterfowl.

There are at least 144 strains of the AI virus, organized into two groups: the first group, based on hemagglutinin proteins, includes 16 types (H1–H16); the second group, based on neuraminidase proteins, includes 9 types (N1–N9).¹

All AI strains can also be characterized as either low pathogenic AI (LPAI) or highly pathogenic AI (HPAI). Most AI strains are low pathogenic and cause only mild or asymptomatic infections. However, the HPAI strains are highly infectious and cause severe illness that can result in 90–100% mortality of the flock.

Signs and Diagnosis
Signs of AI in poultry can include inappetence and depression; diarrhea; a drop in egg production; soft-shelled or misshapen eggs; cyanosis and edema of the head, comb and wattle; sneezing; coughing; and bloodstained nasal and oral discharges. LPAI signs are mild, and the disease may be asymptomatic. HPAI signs are severe; in some cases, HPAI can cause sudden death with no clinical signs.

A definitive AI diagnosis requires isolation and identification of the virus.

Transmission
AI is transmitted by contact with feces and secretions from infected birds; broken infected eggs; and contaminated feed, water, equipment and clothing. Humans can spread the disease among flocks after contact with contaminated birds and materials. Wild birds can also introduce the disease to domestic poultry populations.

LPAI poses no known risk to humans, but some strains of LPAI can mutate into the highly pathogenic forms. A few strains of HPAI, in particular the H5N1 strain, can be passed from poultry to humans. Mortality rates for human infections are approximately 60%.²

AI infections are monitored closely worldwide to detect any spread to human populations. There is concern that the virus may eventually mutate to a form that can be transmitted from person to person.

Disease Monitoring
Because of the wide variety and severity of clinical AI symptoms, serological testing is used to accurately detect the disease. Monitoring the exposure of a flock to influenza viruses is accomplished by measuring the presence and amount of AI antibody in serum. Regular monitoring is essential to detect and prevent the spread of AI.

The new IDEXX FlockChek* AI MultiS-Screen Ab test is a highly specific and sensitive enzyme-linked immunosorbent assay (ELISA) for monitoring AI exposure in multiple avian species, including chickens, turkeys, ducks, ostrich, geese. The assay does not require species-specific reagents.

References

1. US Department of Agriculture Animal and Plant Health Inspection Service. Audit Report No. 33099-11-Hy. June 2006.
2. Centers for Disease Control and Prevention. Avian influenza: current H5N1 situation. Available at www.cdc.gov/flu/avian/outbreaks/current.htm. Accessed January 8, 2008.

Introducing the IDEXX FlockChek* Avian Influenza
MultiS-Screen Ab Test Kit

The new IDEXX FlockChek* Avian Influenza MultiS-Screen Ab Test Kit tests for avian influenza (AI) in one step for multiple species, with 99.7% specificity and detection at 13 days postinfection.

Using the new FlockChek AI MultiS-Screen Ab test, your laboratory can:

• Run fewer confirmatory tests—near 100% specificity means fewer false-positive samples that require additional testing.
• Detect AI antibodies even when the infecting subtype is unknown. The FlockChek AI MultiS-Screen Ab test performs as well as homologous hemagglutination inhibition (HI) tests, regardless of AI subtype.
• Improve laboratory throughput by assaying more than one species at a time, using the same reagents and protocol. There is no need to maintain multiple kits with different expiration dates and storage requirements.

IDEXX has validated the FlockChek AI MultiS-Screen Ab test with chicken, turkey, duck, ostrich and goose samples. The test demonstrated specificity approaching 100% for all tested species. It detected positives at the same point as agar gel immunodiffusion (AGID) tests at 13 days postinfection and equal to homologous HI, without subtype limitations. Overall, the FlockChek AI MultiS-Screen Ab test demonstrated an excellent balance between sensitivity and specificity.

Validation methods are described below.

Multispecies Specificity
Serum samples from more than 5,000 chickens, turkeys, ducks, geese and ostrich from various countries were validated using the FlockChek AI MultiS-Screen Ab Test Kit. Samples included sera from nonvaccinated, specific-pathogen-free (SPF) birds and from birds highly vaccinated against non-AI infectious agents. Prior to validation, all samples were verified to be AI-negative by reference tests or by the IDEXX indirect AI ELISA test.

Conclusion—The overall specificity of the AI MultiS-Screen Ab test was 99.7%.

Sensitivity Compared to AGID Tests
Three groups of SPF chickens were inoculated with influenza vaccines, each for a different AI subtype (H1N1, H5N2, and H7N3). Sera were collected and pooled from birds in each group on each bleed date. Samples were tested using AI MultiS-Screen ELISA and AGID.

Conclusion—The FlockChek AI MultiS-Screen Ab test detected all three AI subtypes at 13 days postinfection, comparable to the AGID test results.

Sensitivity Compared to HI Tests
Chicken and duck serum samples were tested using HI, then tested again using the FlockChek AI MultiS-Screen Ab test.

Four groups of chicken samples were tested from populations vaccinated or exposed (or both) to the H5N2 subtype; the associated HI test was homologous to the subtype.

Duck samples included six groups, each vaccinated with one of four subtypes (or a combination); the associated HI tests were homologous to two of the subtypes.

Results from the two test methods were compared.

Results—The results for chicken samples were comparable to HI results; all samples that were called positive by HI were also called positive by the FlockChek AI MultiS-Screen Ab test. For duck samples, the FlockChek AI MultiS-Screen Ab test detected antibodies in all six vaccinated duck groups, whereas HI detected positives only when the vaccine subtype matched the HI subtype.

Conclusion—The sensitivity of the FlockChek AI MultiS-Screen Ab test matched or exceeded that of HI, but without HI subtype limitations.

Summary
The FlockChek AI MultiS-Screen Ab test:

• Has an overall specificity of 99.7% and 95.4% sensitivity
• Detects AI antibodies at 13 days postinfection, comparable to AGID tests
• Performs as well as homologous HI testing, without HI subtype limitations
• Successfully detects AI antibodies across multiple avian species
• Excellent specificity without compromising sensitivity

The FlockChek AI MultiS-Screen Ab test is an accurate and reliable tool for AI screening of multiple avian species.

Compare the New IDEXX FlockChek AI MultiS-Screen Ab Test to the IDEXX AI ELISA

There are some differences between the new IDEXX FlockChek* AI MultiS-Screen Ab test and its predecessor, the IDEXX AI ELISA. The differences are outlined below.

Kit Components

FlockChek AI MultiS-Screen Ab Test Kit

• 5 plates (solid format)
• Positive control, negative control
• Anti-AI HRPO conjugate
   
• Sample diluent
• TMB substrate
• Stop solution
• Wash concentrate
   

Kit Protocols
The easy-to-learn protocol is similar to that for the IDEXX AI ELISA and is a blocking assay protocol identical to the one for the FlockChek Chicken Anemia Virus Ab test.

Results Interpretation
The FlockChek AI MultiS-Screen Ab test calculates the ratio of the sample value to the negative control value (S/N), instead of the sample value to the positive control value (S/P). Color development in the new test indicates a negative result, rather than a positive result.

Events Around the World

• AgroFarm Exhibition RU – Moscow—April 24–26, 2008
  www.agrofarm.org
• Avesui 2008 - Florianopolis, BR—May 13–15, 2008
  www.avesui.com
• Apinco - Santos, Brazil—May 27–29, 2008
  www.facta.org.br
• National Poultry Improvement Plan - Portland Me.—June 4–7, 2008
  www.wpc2008.com

Use xChek* Software to Analyze Data from the FlockChek* AI MultiS-Screen Ab Test Kit

When a new test, such as the FlockChek* AI MultiS-Screen Ab test, is introduced, you can easily add it to your xChek software. This process is called creating a user-defined assay (UDA). Step-by-step instructions for adding a UDA to the software can be found at idexx.com on the xChek software page. The UDA allows you to use xChek software, available from IDEXX Laboratories, to run the test on a plate reader and to store and analyze the data generated from the test.

For user-defined assays, xChek does not evaluate controls to see whether they meet the assay validity specifications outlined in the test kit insert. You must evaluate the controls from each plate yourself, as outlined in the product insert, to determine whether the assay is valid. If you have questions about UDAs, please contact your area manager or local technical service representative.

IDEXX welcomes Tony Carter—Area Sales Manager, PAS

IDEXX Laboratories is pleased to announce Tony Carter has joined Production Animal Services. In his role as Area Sales Manager for PAS, Tony will manage all sales of livestock and poultry products for IDEXX and Institut Pourquier in the UK and Ireland. Most recently, he served as a Veterinary Products Manager at Guildhay Ltd. and has gained broad experience in various clinical diseases, ELISA development, and technical sales.

Tony is married and has three sons. He and his family live north of London, and his interests are cooking, rugby and walking holidays.

Tony’s contact information is:

Office: +44 1234 713783
Mobile: +44 7500 014809
Fax: +44 1234 713783

E-mail: Tony-Carter@IDEXX.com

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