| 1. |
- Kraus, Robert H. S., et al.
(författare)
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Avian influenza surveillance with FTA cards : field methods, biosafety, and transportation issues solved
- 2011
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Ingår i: Journal of Visualized Experiments. - : MyJove Corporation. - 1940-087X. ; :54
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Tidskriftsartikel (refereegranskat)abstract
- Avian Influenza Viruses (AIVs) infect many mammals, including humans(1). These AIVs are diverse in their natural hosts, harboring almost all possible viral subtypes(2). Human pandemics of flu originally stem from AIVs(3). 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 strains(5), dispersal can also partly be attributed to wild birds(6, 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 strains(8-12). 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 recognised(13, 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 cards(19) preserve RNA on a dry storage basis(20) and render pathogens inactive upon contact(21). 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.
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| 2. |
- Kraus, Robert H. S., et al.
(författare)
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Genome wide SNP discovery, analysis and evaluation in mallard (Anas platyrhynchos)
- 2011
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Ingår i: BMC Genomics. - 1471-2164 .- 1471-2164. ; 12, s. 150-
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Tidskriftsartikel (refereegranskat)abstract
- Background: Next generation sequencing technologies allow to obtain at low cost the genomic sequence information that currently lacks for most economically and ecologically important organisms. For the mallard duck genomic data is limited. The mallard is, besides a species of large agricultural and societal importance, also the focal species when it comes to long distance dispersal of Avian Influenza. For large scale identification of SNPs we performed Illumina sequencing of wild mallard DNA and compared our data with ongoing genome and EST sequencing of domesticated conspecifics. This is the first study of its kind for waterfowl. Results: More than one billion base pairs of sequence information were generated resulting in a 16x coverage of a reduced representation library of the mallard genome. Sequence reads were aligned to a draft domesticated duck reference genome and allowed for the detection of over 122,000 SNPs within our mallard sequence dataset. In addition, almost 62,000 nucleotide positions on the domesticated duck reference showed a different nucleotide compared to wild mallard. Approximately 20,000 SNPs identified within our data were shared with SNPs identified in the sequenced domestic duck or in EST sequencing projects. The shared SNPs were considered to be highly reliable and were used to benchmark non-shared SNPs for quality. Genotyping of a representative sample of 364 SNPs resulted in a SNP conversion rate of 99.7%. The correlation of the minor allele count and observed minor allele frequency in the SNP discovery pool was 0.72. Conclusion: We identified almost 150,000 SNPs in wild mallards that will likely yield good results in genotyping. Of these, similar to 101,000 SNPs were detected within our wild mallard sequences and similar to 49,000 were detected between wild and domesticated duck data. In the similar to 101,000 SNPs we found a subset of similar to 20,000 SNPs shared between wild mallards and the sequenced domesticated duck suggesting a low genetic divergence. Comparison of quality metrics between the total SNP set (122,000 + 62,000 = 184,000 SNPs) and the validated subset shows similar characteristics for both sets. This indicates that we have detected a large amount (similar to 150,000) of accurately inferred mallard SNPs, which will benefit bird evolutionary studies, ecological studies (e. g. disentangling migratory connectivity) and industrial breeding programs.
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| 3. |
- Kraus, Robert H. S., et al.
(författare)
-
Genome wide SNP discovery, analysis and evaluation in mallard (Anas platyrhynchos)
- 2011
-
Ingår i: BMC Genomics. - : BioMed Central Ltd.. - 1471-2164. ; 12
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Next generation sequencing technologies allow to obtain at low cost the genomic sequence information that currently lacks for most economically and ecologically important organisms. For the mallard duck genomic data is limited. The mallard is, besides a species of large agricultural and societal importance, also the focal species when it comes to long distance dispersal of Avian Influenza. For large scale identification of SNPs we performed Illumina sequencing of wild mallard DNA and compared our data with ongoing genome and EST sequencing of domesticated conspecifics. This is the first study of its kind for waterfowl. Results: More than one billion base pairs of sequence information were generated resulting in a 16x coverage of a reduced representation library of the mallard genome. Sequence reads were aligned to a draft domesticated duck reference genome and allowed for the detection of over 122,000 SNPs within our mallard sequence dataset. In addition, almost 62,000 nucleotide positions on the domesticated duck reference showed a different nucleotide compared to wild mallard. Approximately 20,000 SNPs identified within our data were shared with SNPs identified in the sequenced domestic duck or in EST sequencing projects. The shared SNPs were considered to be highly reliable and were used to benchmark non-shared SNPs for quality. Genotyping of a representative sample of 364 SNPs resulted in a SNP conversion rate of 99.7%. The correlation of the minor allele count and observed minor allele frequency in the SNP discovery pool was 0.72. Conclusion: We identified almost 150,000 SNPs in wild mallards that will likely yield good results in genotyping. Of these, similar to 101,000 SNPs were detected within our wild mallard sequences and similar to 49,000 were detected between wild and domesticated duck data. In the similar to 101,000 SNPs we found a subset of similar to 20,000 SNPs shared between wild mallards and the sequenced domesticated duck suggesting a low genetic divergence. Comparison of quality metrics between the total SNP set (122,000 + 62,000 = 184,000 SNPs) and the validated subset shows similar characteristics for both sets. This indicates that we have detected a large amount (similar to 150,000) of accurately inferred mallard SNPs, which will benefit bird evolutionary studies, ecological studies (e. g. disentangling migratory connectivity) and industrial breeding programs.
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