We wish to thank M. Scullion and R. Crisp of Idaho Technologies for technical support and the USGS Western Ecological Research Center for 
funding (S. Schwarzbach) and assistance (K. Spragens, T. Graham). This research was performed under the auspices of the Center for Innovative Technology - 
Institute for Defense and Homeland Security (www.idhs.org), in support of the Department of Defense and Air Force Research Laboratory. 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. Any use of trade, product, or firm names in this publication is for descriptive purposes only and does not imply endorsement by the 
U. S. government.

1. Wild bird capture using mist nets
<ol>
<li>For shorebird capture, set up mist nets at an active foraging site such as a marsh, shoreline, or mud flat.</li>
<li>Slide trammel line loops of one end of the mist net around the pole and insert pole vertically into mud.</li>
<li>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.</li>
<li>Once a bird is captured, extract the bird from the net and return to t...

<ol>
	<li>Spackman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+real-time+reverse+transcriptase+PCR+assay+for+type+A+influenza+virus+and+the+avian+H5+and+H7+hemagglutinin+subtypes.">Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes.</a> J Clin Microbiol. 40, 3256-3260 (2002).</li><li>Takekawa, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Field+detection+of+avian+influenza+virus+in+wild+birds:+evaluation+of+a+portable+rRT-PCR+system+and+freeze-dried+reagents.">Field detection of avian influenza virus in wild birds: evaluation of a portable rRT-PCR system and freeze-dried reagents.</a> J Virol Methods. 166, 92-97 (2010).</li><li>Das, A., Spackman, E., Senne, D., Pedersen, J., Suarez, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+internal+positive+control+for+rapid+diagnosis+of+avian+influenza+virus+infections+by+real-time+reverse+transcription-PCR+with+lyophilized+reagents.">Development of an internal positive control for rapid diagnosis of avian influenza virus infections by real-time reverse transcription-PCR with lyophilized reagents.</a> J Clin Microbiol. 44, 3065-3073 (2006).</li><li>Spackman, E., Suarez, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avian+influenza+virus+RNA+extraction+from+tissue+and+swab+material.">Avian influenza virus RNA extraction from tissue and swab material.</a> Methods Mol Biol. 436, 13-18 (2008).</li><li>Chen, R., Holmes, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequent+inter-species+transmission+and+geographic+subdivision+in+avian+influenza+viruses+from+wild+birds.+Virology.">Frequent inter-species transmission and geographic subdivision in avian influenza viruses from wild birds. Virology.</a> , 383-3156 (2009).</li><li>Macken, C. A., Webby, R. J., Bruno, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotype+turnover+by+reassortment+of+replication+complex+genes+from+avian+influenza+A+virus.">Genotype turnover by reassortment of replication complex genes from avian influenza A virus.</a> J Gen Virol. 87, 2803-2815 (2006).</li><li>Dugan, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolutionary+genetics+and+emergence+of+avian+influenza+viruses+in+wild+birds.">The evolutionary genetics and emergence of avian influenza viruses in wild birds.</a> PLoS Pathog.. 4, e1000076-e1000076 (2008).</li><li>Spackman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+analyses+of+type+A+influenza+genes+in+natural+reservoir+species+in+North+America+reveals+genetic+variation.">Phylogenetic analyses of type A influenza genes in natural reservoir species in North America reveals genetic variation.</a> Virus Res. 114, 89-100 (2005).</li><li>Pasick, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+molecular+based+techniques+for+the+diagnosis+and+characterization+of+avian+influenza+virus+infections..">Advances in the molecular based techniques for the diagnosis and characterization of avian influenza virus infections..</a> Transbound Emerg Dis. 55, 329-338 (2008).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OIE+Manual+of+diagnostics+tests+and+vaccines+for+terrestrial+animals+(mammals,+birds+and+bees).">OIE Manual of diagnostics tests and vaccines for terrestrial animals (mammals, birds and bees).</a> 1, 258-269 (2004).</li><li>Weber, T. P., Stilianakis, N. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecologic+immunology+of+avian+influenza+(H5N1)+in+migratory+birds.">Ecologic immunology of avian influenza (H5N1) in migratory birds.</a> Emerg. Infect. Dis. 13, 1139-1143 (2007).</li><li>Kim, J. -. K., Negovetich, N. J., Forrest, H. L., Webster, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ducks:+the+"Trojan+Horses"+of+H5N1+influenza.">Ducks: the "Trojan Horses" of H5N1 influenza.</a> Influenza and Other Respiratory Viruses. , 121-128 (2009).</li><li>Gilbert, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flying+over+an+infected+landscape:+distribution+of+highly+pathogenic+avian+influenza+H5N1+risk+in+South+Asia+and+satellite+tracking+of+wild+waterfowl.">Flying over an infected landscape: distribution of highly pathogenic avian influenza H5N1 risk in South Asia and satellite tracking of wild waterfowl.</a> Ecohealth. , (2010).</li></ol>

No conflicts of interest declared.

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...

<table border="1">
<tbody>
 <tr>
 <th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue Number</th>
 <th>Comments (optional)</th>
 </tr>
 <tr>
 <td>RNeasy mini spin column</td>
 <td>Qiagen</td>
 <td>74106</td>
 <td>Included in RNeasy Mini Kit</td>
 </tr>
 <tr>
 <td>Collection tubes (1.5 &amp; 2 mL)</td>
 <td>Qiagen</td>
 <td>74106</td>
 <td>Included in RNeasy Mini Kit</td>
 </tr>
 <tr>
 <td>Buffer RLT</td>
 <td>Qiagen</td>
 <td>74106</td>
 <td>Included in RNeasy Mini Kit</td>
 </tr>
 <tr>
 <td>Buffer RW1</td>
 <td>Qiagen</td>
 <td>74106</td>
 <td>Included in RNeasy Mini Kit</td>
 </tr>
 <tr>
 <td>Buffer RPE</td>
 <td>Qiagen</td>
 <td>74106</td>
 <td>Included in RNeasy Mini Kit</td>
 </tr>
 <tr>
 <td>RNase-free water</td>
 <td>Qiagen</td>
 <td>74106</td>
 <td>Included in RNeasy Mini Kit</td>
 </tr>
 <tr>
 <td>14.3 M &beta;-mercaptoethanol solution</td>
 <td>Fisher Scientific</td>
 <td>BP176100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>100% ethanol</td>
 <td>Fisher Scientific</td>
 <td>NC9602322</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Vortex Genie 2, 120V</td>
 <td>Scientific Industries</td>
 <td>SI-0236</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Taqman Influenza A Target 1 (Hydrolysis Probe)</td>
 <td>Idaho Technologies</td>
 <td>ASAY-ASY-0109</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Lightcycler 20ml capillary tubes</td>
 <td>Roche Applied Science</td>
 <td>04929292001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Micro-centrifuge with rotator for 2 ml tubes </td>
 <td>Idaho Technologies</td>
 <td>&nbsp;</td>
 <td>Included in RAPID kit</td>
 </tr>
 <tr>
 <td>Ruggedized Advanced Pathogen Identification Device (RAPID) 7200</td>
 <td>Idaho Technologies</td>
 <td>&nbsp;</td>
 <td>Included in RAPID kit</td>
 </tr>
 <tr>
 <td>Pentium-based laptop with Windows XP Professional</td>
 <td>Idaho Technologies</td>
 <td>&nbsp;</td>
 <td>Included in RAPID kit</td>
 </tr>
 <tr>
 <td>Lightcycler Data Analysis software</td>
 <td>Idaho Technologies</td>
 <td>&nbsp;</td>
 <td>Included in RAPID kit</td>
 </tr>
 </tbody>
</table>

rapid diagnosis of avian influenza virus in wild birds: use of a portable rrt-pcr and freeze-dried reagents in the field

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.

Watch this Scientific Journal Video about Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field at JoVE.com

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

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.

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory ...

rapid-diagnosis-avian-influenza-virus-wild-birds-use-portable-rrt-pcr

Research

JoVE Journal

Immunology and Infection

15.6K Görüntüleme.  USGS Western Ecological Research Center. Bu çalışmada, taşınabilir bir RRT-PCR sistemi kullanarak vahşi kuşlarda kuş gribi teşhisi açıklamaktadır. Bu yöntem, bir salgın senaryosunun tipik olmayan bir laboratuar ortamında yabani kuşlar, ekrana dondurularak kurutulmuş reaktifler yararlanır. Moleküler araçlarını kullanın, hızlı tanı için doğru ve hassas alternatifler sunuyor.