Georgia Veterinary Scholars Program at the UGA College of Veterinary Medicine

The University of Georgia College of Veterinary Medicine

2005 Georgia Veterinary Scholars

Georgia Veterinary Scholar	Faculty Mentor

Bridget Fitzpatrick Tuskegee University Class of 2007	Dr. David Suarez

Development of a Real-Time Reverse Transcription Polymerase Chain Reaction Assay for Avian Influenza Hemagglutinin Subtype H12

Bridget Fitzpatrick*, David L. Suarez, Erica Spackman.

Rapid, cost-effective epidemiologic surveillance of all subtypes of avian influenza is critical in efforts to identify and control outbreaks and thereby protect our economic and trade interests. A real-time reverse transcription polymerase chain reaction (RRT-PCR) assay exists for the detection of all Type A influenza virus, and additional tests are available to identify avian hemagglutinin subtypes H5 and H7. This RRT-PCR assay is of comparable efficacy (89%), significantly faster, and more economical then the gold standard of virus isolation (Spackman et al, 2002). However, RRT-PCR tests are not available for many of the other subtypes. The hemagglutinin subtype H12 is a low pathogenic variety of avian influenza (AI), but it has caused outbreaks of disease in poultry in the U.S. A similar RRT-PCR assay was sought and evaluated in this project for hemagglutinin subtype H12.

The assay was developed by designing two primer/probe sets based on conserved regions in the genome of six North American AI H12 isolates. These sets were evaluated for sensitivity and specificity utilizing samples from four isolates in the RRT-PCR reaction. Comparing the two primer/probe sets showed one to be more sensitive for all four isolates of H12 assessed (up to 10^-6 and 10^-7 dilutions), but its specificity was limited by detection of hemagglutinin subtypes six and fourteen at RRT-PCR cycles of 30-32. The other probe set, while specific for H12, had poor sensitivity to two of the H12 isolates (up to 10^-3 dilution). Both primer/probe sets were optimized for sensitivity by comparing various annealing temperatures, forward: reverse primer ratios, and probe and magnesium chloride concentrations.

In order to evaluate the field efficacy of the developed H12 RRT-PCR assay, seven day-old specific pathogen free white leghorn chickens were intranasally inoculated with two H12 AIV isolates. The groups of sixteen and fifteen birds, were inoculated with H12 isolates RTS/DE/650644/02 and RTS/A100-82819/01 respectively, and were swabbed daily, by oral and cloacal routes on days 0, 2, 3, and 6 post-inoculation. Future research should analyze the applicability of this test for detection of H12 AI in live-bird markets and compare its efficacy to that of virus inoculation in terms of sensitivity and specificity. Additional probe sets might be necessary in order to develop a more sensitive and specific assay. It is proposed that the developed assay will provide the advantages of speed, minimized potential of cross-contamination, and ease of use over the commonly used virus isolation technique in detection of H12 avian influenza.