Using swabs collected from chickens experimentally exposed to LPAIV we evaluated numerous elements of sample collection and transport for AIV and evaluated swab pooling with NDV. This data provides concrete information on which factors in sample collection are critical for the most common tests for AIV. Thus allowing surveillance programs to be better tailored to specific situations, including what the best alternatives are when some component of an optimal program is not available.
Based on this data, the optimal method for collecting AIV oral swab samples from poultry would be flocked swabs with BHI media as this provided the best results consistently with all three detection methods. The results with the AgIA were complicated, as there was variation based on assay, swab construction type and media, but the most positives were observed consistently with flocked swabs.
Previous studies  have shown the superiority of BHI to PBS for the isolation of AIV, but this had not been evaluated using rRT-PCR, a more commonly used surveillance tool at the present time. The improved capture and release characteristics of flocked swabs have been shown with clinical specimens previously [6, 8, 9] and one study found that foam swabs were superior to flocked swabs for rapid influenza testing using human specimens , which is consistent with our results. Flocked swabs were marginally better than foam, but not always significantly. Therefore, foam swabs could be used if the cost (currently the cost is similar) or availability of flocked swabs prevents their use. Also, while the reasons are not clear at this time, foam swabs should not be used in conjunction with PBS media for AgIA testing since results using both kits were poor. Regarding media, in certain situations where only rRT-PCR will be used and VI will not be attempted (which is rare), PBS which is cheaper and has a longer shelf life at refrigeration temperatures may be used as a transport media.
Transporting the specimen with wet media was most critical for VI, but also seemed to affect detection of AIV by rRT-PCR as titers were reduced. This seemed to contradict a previous study where wet and dry transport of swabs from ducks was compared , however the differences in results may be explained by a few methodological differences. The key difference was that virus isolation was not attempted, and this is where the differences were most clear in this study. Furthermore, their samples were from Pekin ducks, which primarily shed cloacally and ours were from chickens, therefore as Roelandt, et a. suggest, the fecal material may protect the virus , which we could not evaluate since there were too few positive cloacal swab samples from chickens.
Additionally, it did not matter whether the swab was left in the media during transport or wrung out and removed prior to transport. It should be taken into account that when multiple swabs are left in the media they will absorb it, which reduces the amount available for testing. The advantage of removing the swab prior to transport is that it eases processing for the diagnostic lab, eliminates the loss of media due to absorption by the swabs, and reduces the potential for cross-contamination by aerosols in the event that the swabs are removed in the lab.
Media volume was evaluated because it can have a direct effect on virus concentration and the concentration of inhibitors in a specimen. The initial volume used in a swab tube needs to balance media cost with having adequate final volume to conduct all necessary tests. In addition the initial volume and tube size must allow all swabs to be immersed in the transport media in the event the swabs are left in the tube. The initial volume also needs to account for the number swabs which may be collected in a single vial, since more swabs will absorb more media regardless of how well the material is expressed from the swab. For example a vial in which 11 swabs are collected must have more volume (minimum 5.5 ml) than a vial in which only 5 are collected (minimum of about 3.5 ml). There are two aspects to this question: 1) what should the initial volume be? And 2) if fewer swabs are collected in a vial prepared with a volume for a larger amount will this affect detection? Fortunately initial volume did not have a measureable effect on any of the detection assays with OP swabs.
Swab pooling is used to decrease costs by consolidating the samples from a single premise or flock. Five swabs per vial has frequently been used as a maximum recommended number. An upper limit of 11 swabs has been suggested based on the statistical calculation where 11 swabs from a flock (of 10,000 or more) should be sufficient to detect 25% infection rate with 95% confidence, which is the level of surveillance outlined by the NPIP. A previous study comparing 5 and 11 swabs for detection of LPAIV from broilers by rRT-PCR has been reported and found no difference . Another study comparing a single swab with pools of 5 with samples from turkeys also found no difference  This study agrees with those results and adds a comparison with single swabs, five and 11 swab pools in one experiment and very importantly, data for VI. Furthermore, NDV was included in the evaluation of swab pooling because it is an important differential of AIV and pooled specimens are often tested for both AIV and NDV. Swab pooling from NDV infected birds provided similar results, indicating that pooling up to 11 swabs would not affect detection by rRT-PCR or VI.
Although the detection data seems to support that up to 11 swabs can be pooled in a vial, it is critical to note that when implementing testing programs logistics for the field and diagnostic lab must be considered. In this case, a larger vial is needed to assure all swabs are immersed in the transport media if the swabs are going to left in it during transport, and to accommodate an increase in media volume to assure sufficient media is present for both surveillance and confirmation testing which includes virus isolation. The larger 11 swab vial is more difficult to handle, thus increasing the time to process. Also with all the samples in one tube if loss or breakage occurs new samples must be collected. The entire testing process needs to be considered in the cost-benefit assessment of any testing program.
As expected, the differences observed were relatively minor since the detection tests are already so near to their limits of detection, although a few parameters were statistically significant. In addition to providing data on what the optimal methods are, this work also showed that there are some sample collection and transport methods which should be avoided, for example using foam swabs with PBS and transporting swabs dry. BHI was shown to be the optimal vial transport media especially when specimens are collected after 3 days post exposure.