1Resource Ecology Group, Wageningen University, 2Section for Zoonotic Ecology and Epidemiology, School of Natural Sciences, Linnaeus University, 3Centre for Wildlife Ecology, Simon Fraser University
Kraus, R. H., van Hooft, P., Waldenström, J., Latorre-Margalef, N., Ydenberg, R. C., Prins, H. H. Avian Influenza Surveillance with FTA Cards: Field Methods, Biosafety, and Transportation Issues Solved. J. Vis. Exp. (54), e2832, doi:10.3791/2832 (2011).
Avian Influenza Viruses (AIVs) infect many mammals, including humans1. These AIVs are diverse in their natural hosts, harboring almost all possible viral subtypes2. Human pandemics of flu originally stem from AIVs3. Many fatal human cases during the H5N1 outbreaks in recent years were reported. Lately, a new AIV related strain swept through the human population, causing the 'swine flu epidemic'4. Although human trading and transportation activity seems to be responsible for the spread of highly pathogenic strains5, dispersal can also partly be attributed to wild birds6, 7. However, the actual reservoir of all AIV strains is wild birds.
In reaction to this and in face of severe commercial losses in the poultry industry, large surveillance programs have been implemented globally to collect information on the ecology of AIVs, and to install early warning systems to detect certain highly pathogenic strains8-12. Traditional virological methods require viruses to be intact and cultivated before analysis. This necessitates strict cold chains with deep freezers and heavy biosafety procedures to be in place during transport. Long-term surveillance is therefore usually restricted to a few field stations close to well equipped laboratories. Remote areas cannot be sampled unless logistically cumbersome procedures are implemented. These problems have been recognised13, 14 and the use of alternative storage and transport strategies investigated (alcohols or guanidine)15-17. Recently, Kraus et al.18 introduced a method to collect, store and transport AIV samples, based on a special filter paper. FTA cards19 preserve RNA on a dry storage basis20 and render pathogens inactive upon contact21. This study showed that FTA cards can be used to detect AIV RNA in reverse-transcription PCR and that the resulting cDNA could be sequenced and virus genes and determined.
In the study of Kraus et al.18 a laboratory isolate of AIV was used, and samples were handled individually. In the extension presented here, faecal samples from wild birds from the duck trap at the Ottenby Bird Observatory (SE Sweden) were tested directly to illustrate the usefulness of the methods under field conditions. Catching of ducks and sample collection by cloacal swabs is demonstrated. The current protocol includes up-scaling of the work flow from single tube handling to a 96-well design. Although less sensitive than the traditional methods, the method of FTA cards provides an excellent supplement to large surveillance schemes. It allows collection and analysis of samples from anywhere in the world, without the need to maintaining a cool chain or safety regulations with respect to shipping of hazardous reagents, such as alcohol or guanidine.
1. Duck Trapping and Cloacal Swabbing
2. Viral RNA Isolation
3. RT-PCR of the AIV Matrix Gene
Carry out reverse transcription PCR (RT-PCR) with the One-Step Access RT-PCR system (Promega) in 25 μl reactions (adjusted from Kraus et al.18 and Fouchier et al.22):
|Nuclease free water||1.5μl|
|primer M52C22 (10 μM)||2.5μl|
|primer M253R22 (10 μM)||2.5μl|
|MgSO4 (25 mM)||7μl|
|AMV reverse transcriptase (5u/μl)||0.5μl|
|Tfl polymerase (5u/μl)||0.5μl|
PCR conditions in a Biometra T1 thermocycler are: initial reverse transcription of 45 minutes at 45°C, followed by 2 minutes initial denaturation at 94°C and 40 cycles of: 94°C for 1 minute, 56°C for 1 minute, and 68°C for 2 minutes. An additional 7 minutes elongation at 68°C concludes the amplification.
4. Screening for AI-positive Samples and Purification of Targeted Fragments from Gel
5. Sequencing and Identification of PCR Products
6. Representative Results:
Mallards (Anas platyrhynchos) were sampled at Ottenby Bird Observatory in December 2007. From each mallard a sample on FTA card was taken as described in this protocol. After shipping, the FTA cards were kept in a freezer at -20°C for two years. The same FTA card sample of the laboratory isolate tested in Kraus et al.18 was included as positive control, as well as nine tenfold serial dilutions of it. Two negative controls were i) extraction from an empty FTA card, to test if there was carry-over from the puncher, and ii) RT-PCR reaction in which nuclease free water was used as template, to test if contamination occurred during, or in preparation of the PCR reaction.
84 samples were analysed. A gel picture of the PCR products from these 84 samples can be found in Figure 1. From natural samples a multitude of unspecific bands can be observed due to the presence of various microbial contaminations in the faeces. However, the target fragment of the primer pair is 244 bp long. The whole PCR-reaction volume of a subset of the samples which produced fragments in approximately the correct size range (between 200 bp and 300 bp) was loaded on gel (Figure 1). An illustration of which of the bands were cut from the gel can be found in Supplementary Figure 1. In addition to the positive control, two of these samples (69899 and 69912) were positive by the new protocol. A BLAST search against the NCBI nucleotide database revealed their identity as AI matrix gene (Figure 3), while all the other bands resembled bacterial sequences most closely, or did not yield a readable sequence at all.
Figure 1. Gel picture of a preliminary screening of the PCR products. 2 μl PCR product of positive control, serial dilutions of the positive control, two negative controls and 84 cloacal samples were loaded. 48 samples are shown in the top gel panel, and 48 samples in the bottom panel. Red arrows indicate gel lanes used for 100 bp DNA size standard. For illustration, red circles show bands in the expected size range. Blue circles indicate an unspecific amplicon (top left) or a primer dimer artefact below 100 bp in size (bottom right).
Figure 2. Selection of samples with fragments in the correct size range. Samples with bands between 200 bp and 300 bp (target fragment 244 bp) were chosen. The green arrow indicates the positive control, the two red arrows indicate samples which were confirmed to be AIV positive by comparing their cDNA sequences to the NCBI nucleotide database.
Figure 3. Screen capture of a representative BLAST search at NCBI. One of the cDNA sequences obtained from the excised fragments was queried against the nucleotide database at the NCBI website. The sample is correctly identified as AI Matrix gene fragment. To view a full sized version of this image, click here.
Supplementary Figure S1. Bands of selected samples excised from gel. This picture was taken from the gel depicted in Figure 1 after candidate bands between 200 bp and 300 bp were cut out.
The protocol described here provides a supplementary method to screen faecal or similar samples for the presence of AIV. It was especially designed to make sample collection quick and easy. This makes it possible for less trained persons, such as hunters or wildlife managers, to contribute to AI surveillance. No cool chains need to be applied, although freezing the samples is recommended where possible. A few days of room temperature, for instance during transport to the laboratory, were not a problem for RNA molecules on FTA cards, as long as the cards remained dry. Other non-cooled storage systems that are currently evaluated by the research community, such as alcohol15 or guanidine16, require special shipping arrangements because of their hazardous nature. In contrast, the FTA card method does not require shipping of hazardous materials. However, another interesting storage medium in this respect is RNAlater™ that is not hazardous, either17. Sample analysis can be carried out in any standard molecular laboratory. No special equipment other than for usual PCR reactions and capillary sequencing was needed. All steps could be carried out without a biosafety level because already at sample collection the potential pathogenic agents were inactivated by the antibacterial and antiviral activity of the FTA card.
Cross-contamination and subsequent false positive samples were not observed in our trials with wild bird samples. However, when working with RNA and PCR it is always advisable to pay special attention to clean working places and separate rooms for pre- and post-PCR steps. Working in a fume hood decreases the risk of aerosol contamination in the laboratory. Pipetting needs to be carried out with filter tips.
From a previous study on samples taken simultaneously from the same ducks we know that six of the 84 samples were positive by the traditional RealTime RT-PCR method24 and the Ct values from RealTime RT-PCR are known. The two positive samples detected by our method stem from ducks which had Ct values <30 (indicating a high concentration of viral RNA). The other four samples which were positive with the traditional protocol had Ct values >30 (less concentrated) and could not be detected by our protocol.
Only samples with relatively high virus titers were positive in our assay and it is likely that sensitivity of the method was the source of failure to detect all positive samples. Further, the sample size in the current study was very low and the method can be completely developed and assessed if more controlled and rigorous experiments are carried out. However, if these samples would have been collected from a remote area, traditional analysis would not have been possible at all. Additionally, these first results stem from pilot experiments which need further optimisation. A period of two years storage in a regular -20°C freezer after sample collection probably also affected the quality of the viral RNA. This possible RNA degradation is an important issue when dealing with room temperature storage of inactivated viruses. Samples stored in alternative liquids as mentioned above suffer from significant degradation which impacts analysis of longer stretches of the viral genome16. Although not tested in our study, FTA cards have proven to be well suited to preserve intact RNA molecules in other RNA systems that are very similar to Avian Influenza viruses25, 26.
Trapping, handling and sampling of birds were done following national legislation and were approved by the Swedish Board of Agriculture and the research animal ethics committee.
We thank Bert Dibbits for technical assistance. The Animal Breeding and Genomics Group, Wageningen University, The Netherlands, generously hosted us in their laboratory. The personnel of the Ottenby Bird Observatory, Sweden, is thanked for trapping and sampling the mallards, in particular Magnus Hellström, Marcus Danielsson, Christopher Magnusson and Stina Andersson. We thank Sanne Svensson, Jonatan Qvist and Per-Axel Gjöres for filming at Ottenby, and Mano Camon for filming in the lab. Daniel Bengtson provided beautiful duck photographs for the video portion of this publication. Further free material from the CDC Public Health Image Library (PHIL; http://phil.cdc.gov/phil/) was used: The electron micrograph (no. 280; by Dr. Erskine Palmer) and illustration (no. 11823; by Douglas Jordan) of the influenza virus. Financial support was given by the KNJV (Royal Netherlands Hunters Association), the Dutch Ministry of Agriculture, the Faunafonds and the Stichting de Eik Trusts (both in The Netherlands), the Swedish Research Council (grant no. 2007-20774) and the EC-founded Newflubird project. RNA isolation chemicals were a generous gift of Ambion, Inc, the RNA company. This is contribution No. 245 from the Ottenby Bird Observatory.
|Plastic rayon dry swab||Copan, Italy||167KS01|
|FTA card||Whatman, GE Healthcare||several options|
|Harris micro punch 2mm with mat||Whatman, GE Healthcare||1154409|
|RNase free 96well plate||Greiner Bio-One||652290||or comparable|
|Kim precision wipes||Kimtech||75512|
|RNA rapid extraction solution||Ambion||AM9775|
|MagMAX-96 viral isolation kit||Ambion||AM1836|
|One-Step Access RT-PCR system||Promega Corp.||A1250||or comparable|
|Agarose MP||Roche Group||1388991001||multi purpose agarose|
|5x loading buffer||Bio-Rad||delivered with DNA ladder|
|EZ load 100 bp ladder||Bio-Rad||170-8353||or comparable|
|Ethidium bromide 1%||Fluka||46067||or comparable|
|1.5ml reaction tubes||Eppendorf||or comparable|
|Zymoclean Gel DNA recovery kit||Zymo Research Corp.||D4001||or comparable|
|ABI big dye 3.1 chemistry||Applied Biosystems||or comparable|