Currently in the US, large numbers of swab samples are collected from domestic poultry and wild birds for avian influenza virus (AIV) detection by real-time RT-PCR (rRT-PCR), commercial antigen immunoassays (AgIA) and virus isolation, according to Erica Spackman of Southeast Poultry Research Laboratory, USDA-Agricultural Research Service in Athens, Georgia.

In a report of research funded by US Poultry & Egg Association, she continued that sample collection procedures are not unified although they are similar for most programmes. Importantly, the specific details of sample collection practices have never been evaluated for their effects on the sensitivity and specificity with rRT-PCR or AgIA, and in some cases, virus isolation.

This work was initiated because Dr Spackman and colleagues identified some new and existing technologies and alternative approaches to swab collection that had the potential to reduce the cost of testing and/or which could improve sensitivity and specificity. Low pathogenicity AIV was the focus of this work because it is diagnostically more difficult to detect than highly pathogenic AIV.

The objective of this work was to determine the optimal sample collection methods for avian influenza virus detection by virus isolation, commercial antigen immunoassays and real-time RT-PCR.

Several aspects of AIV sample collection were evaluated using swabs from experimentally exposed chickens to simulate clinical samples. The three most common diagnostic assays for AIV; rRT-PCR, virus isolation and both commercial AgIA licensed in the US, were used to evaluate the methods.

• Swab type: Three swab types were compared for AIV detection (all cost about $0.12 each currently): non-flocked dacron swabs (current standard in US), nylon-flocked (surface is more like a soft brush) swabs and urethane foam. Of the three swab types, the best performing swab type for AIV detection were the nylon-flocked swabs; and the next was urethane foam. Non-flocked swabs generally performed more poorly and recovered the least virus.
  
  
• Transport media: Brain heart infusion broth (BHI) and phosphate buffered saline (PBS) were compared. Media did not make a difference for rRT-PCR detection. The best media for AgIA was different for non-flocked and foam swabs, whereas flocked swabs performed well with either media and in general better than either other swab type. For virus isolation, BHI was clearly superior to PBS.
  
  
• Swab pooling: There was no difference in virus detection among vials containing one, five or 11 total swabs (one swab from an inoculated bird and the remainder was from unexposed birds). Pooling up to 11 swabs appears to have no effect on virus isolation or rRT-PCR for detection of AIV.
  
  
• Transport conditions: Several combinations of transport conditions were compared: wet transport (3.5ml BHI) with the swab left in the media during transport, wet with the swab removed from the media prior to transport, and swabs transported dry in culturette tubes. Each condition was tested as a single swab and as pools of five swabs. Detection of AIV from BHI by virus isolation was the same whether the swab was left in during transport or removed prior to transport. Transporting dry swabs decreased virus detection by virus isolation substantially. Too few were positive by AgIA to draw conclusions. Based on rRT-PCR, there is a slight advantage to leaving the swab in the vial during transport, however keeping the swab wet was more critical. Virus detection in dry swabs was significantly lower than wet swabs at all time points.
  
  
• Media volume: For pools of five or fewer swabs, lower volumes of media may be used. A volume of 2ml was compared to the standard 3.5ml of BHI. No difference in virus detection was observed for virus isolation but there was marginally better detection by rRT-PCR with 2ml of media.

Inaccurate results of AIV testing (either false negative or false positive) can be very costly for the industry, concluded Dr Spackman. With the large numbers of samples being processed, optimal testing strategies can save money, both by decreasing the cost of initial testing by using less media or by pooling up to 11 swabs per vial.

Pooling more samples could cut pre-slaughter testing costs substantially per flock by reducing the number of tests needed to be run by half and by improving test accuracy.

Finally, Dr Spackman added, this data will save resources in the long term by improving the accuracy of AIV testing by using the optimal swab type (flocked), media (BHI) and transport conditions (wet media) as described above.

Further Reading You can visit the Avian Flu page by clicking here.