The University of Georgia College of Veterinary Medicine

Georgia Veterinary Scholars Program

GVSP Summer 2006 Scholars

Georgia Veterinary Scholar	Faculty Mentor

Azalia Boyd Tuskegee University Class of 2008	Dr. David Suarez

Azalia M. Boyd*, Erica Spackman
Southeast Poultry Research Laboratory, United States Department of Agriculture Research Service, Athens, Georgia

Abstract: Worldwide, there are many strains of avian influenza (AI) virus that cause varying severity of clinical illness in poultry. Low pathogenic AI subtypes have the ability to mutate into more virulent strains. The clinical signs are typically mild to moderate, and can be subclinical. The disease presents primarily as a respiratory infection, however, depression, decreased food consumption, and decreased egg production may occur. Infection in poultry can cause a great economic loss for producers and therefore, a swift, low expenditure diagnostic test of all sixteen subtypes is essential for early detection, control of possible outbreaks, and for surveillance of domestic wild birds. Previously, a real-time reverse transcriptase PCR (RRT-PCR) assay was developed for the rapid detection of type A influenza virus and identifies subtypes from clinical samples. Due to the different hemagglutinin (HA) subtypes described, it is pertinent to be able to detect individual subtypes, distinguishing them from each other. In this experiment, RRT-PCR assay specific for the H14 and H15 HA subtypes were developed and optimized using hydrolysis type probes that were targeted to a conserved region of the H14 and H15 HA genes. The single primer and probe set selected for both the H14 and H15 were optimized comparing concentrations of the forward and reverse primers, probe, and magnesium chloride. Further optimized parameters involved determining the optimal denaturating, annealing, and extension temperatures. Once a positive test result was obtained for the specific subtype, the designed RRT-PCR test was run against 15 different subtypes to test the specificity in identifying the H14 and H15 HA genes. For each of the designed tests, positive results were obtained for the targeted subtype (H14 or H15). The sensitivity of the H14 and H15 RRT-PCR assay were each compared to virus isolation (VI) in embryonating chicken eggs, the reference standard method, using AI virus isolates from Australia (H14) and Russia (H15). The H14 subtype test had a detection limit of 100.75 EID50 and the H15 of 100.5 EID50.