Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field

Summary

This study describes diagnosis of avian influenza in wild birds using a portable rRT-PCR system. The method takes advantage of freeze-dried reagents to screen wild birds in a non-laboratory setting, typical of an outbreak scenario. Use of molecular tools provides accurate and sensitive alternatives for rapid diagnosis.

Abstract

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds for avian influenza virus (AIV) is often conducted in remote regions, but results are often delayed because of the need to transport samples to a laboratory equipped for molecular testing. Real-time reverse transcriptase polymerase chain reaction (rRT-PCR) is a molecular technique that offers one of the most accurate and sensitive methods for diagnosis of AIV. The previously strict lab protocols needed for rRT-PCR are now being adapted for the field. Development of freeze-dried (lyophilized) reagents that do not require cold chain, with sensitivity at the level of wet reagents has brought on-site remote testing to a practical goal.

Here we present a method for the rapid diagnosis of AIV in wild birds using an rRT-PCR unit (Ruggedized Advanced Pathogen Identification Device or RAPID, Idaho Technologies, Salt Lake City, UT) that employs lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies). The reagents contain all of the necessary components for testing at appropriate concentrations in a single tube: primers, probes, enzymes, buffers and internal positive controls, eliminating errors associated with improper storage or handling of wet reagents. The portable unit performs a screen for Influenza A by targeting the matrix gene and yields results in 2-3 hours. Genetic subtyping is also possible with H5 and H7 primer sets that target the hemagglutinin gene.

The system is suitable for use on cloacal and oropharyngeal samples collected from wild birds, as demonstrated here on the migratory shorebird species, the western sandpiper (Calidrus mauri) captured in Northern California. Animal handling followed protocols approved by the Animal Care and Use Committee of the U.S. Geological Survey Western Ecological Research Center and permits of the U.S. Geological Survey Bird Banding Laboratory. The primary advantage of this technique is to expedite diagnosis of wild birds, increasing the chances of containing an outbreak in a remote location. On-site diagnosis would also prove useful for identifying and studying infected individuals in wild populations. The opportunity to collect information on host biology (immunological and physiological response to infection) and spatial ecology (migratory performance of infected birds) will provide insights into the extent to which wild birds can act as vectors for AIV over long distances.

Protocol

1. Wild bird capture using mist nets

1. For shorebird capture, set up mist nets at an active foraging site such as a marsh, shoreline, or mud flat.
2. Slide trammel line loops of one end of the mist net around the pole and insert pole vertically into mud.
3. Stretch out net, insert second pole through trammel loops at other end of mist net and vertically insert pole into mud, making sure that the trammel lines are taught.
4. Once a bird is captured, extract the bird from the net and return to the banding station.

2. Cloacal swab sampling

1. Collect cloacal swabs immediately after capture
2. Lo....

Discussion

The method of rapid diagnosis presented here facilitates time-efficient and accurate testing of wild bird samples for surveillance of AIV. The much less stringent specimen storage requirements of portable rRT-PCR are suitable for remote situations where maintenance of a cold chain may be impractical if liquid nitrogen shippers or dry ice is not available. In addition, we found that sample analysis with freeze-dried reagents was straightforward enough to be performed by field biologists with minimal knowledge of laborato.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RNeasy mini spin column	Qiagen	74106	Included in RNeasy Mini Kit
Collection tubes (1.5 & 2 mL)	Qiagen	74106	Included in RNeasy Mini Kit
Buffer RLT	Qiagen	74106	Included in RNeasy Mini Kit
Buffer RW1	Qiagen	74106	Included in RNeasy Mini Kit
Buffer RPE	Qiagen	74106	Included in RNeasy Mini Kit
RNase-free water	Qiagen	74106	Included in RNeasy Mini Kit
14.3 M β-mercapt–thanol solution	Fisher Scientific	BP176100
100% ethanol	Fisher Scientific	NC9602322
Vortex Genie 2, 120V	Scientific Industries Inc.	SI-0236
Taqman Influenza A Target 1 (Hydrolysis Probe)	Idaho Technologies	ASAY-ASY-0109
Lightcycler 20ml capillary tubes	Roche Group	04929292001
Micro-centrifuge with rotator for 2 ml tubes	Idaho Technologies		Included in RAPID kit
Ruggedized Advanced Pathogen Identification Device (RAPID) 7200	Idaho Technologies		Included in RAPID kit
Pentium-based laptop with Windows XP Professional	Idaho Technologies		Included in RAPID kit
Lightcycler Data Analysis software	Idaho Technologies		Included in RAPID kit