Five 34-wk-old turkey breeder layer flocks in separate houses of 2550 birds each in a single farm in Ohio experienced a drop in egg production from late January to early February 2004. Tracheal swabs (n = 60), cloacal swabs (n = 50), and convalescent sera (n = 110) from the flocks were submitted to the laboratory for diagnostics. Virus isolation was attempted in specific-pathogen free embryonating chicken eggs and Vero and MDCK cells. Virus characterization was performed using agar gel immunodiffusion, the hemagglutination test, the hemagglutination inhibition test, the virus neutralization test, reverse transcription-polymerase chain reaction, sequencing, and phylogenetic analysis. A presumptive influenza virus was successfully propagated and isolated on the first passage in MDCK cells, but initially not in Vero cells or specific-pathogen free chicken embryos. After two passages in MDCK cells, it was possible to propagate the isolate in specific-pathogen free chicken embryos. Preliminary sequence analysis of the isolated virus confirmed that it was influenza A virus with almost 100% (235/236) identity with the matrix gene of a swine influenza A virus, A/Swine/Illinois/100084/01 (H1N2). However, it was not possible to subtype the virus using conventional serotyping methods. The results of genetic characterization of the isolated virus showed that it was the H3N2 subtype and was designated as A/Turkey/OH/313053/04 (H3N2). Phylogenetic analysis of the eight gene segments of the virus showed that A/Turkey/OH/313053/04 (H3N2) isolate was most closely related to the triple-reassortant H3N2 swine viruses [A/Swine/WI/14094/99 (H3N2)] that have been circulating among pigs in the United States since 1998, which contains gene segments from avian, swine, and human viruses. The A/Turkey/OH/313053/04 (H3N2) isolated from turkeys in this study was classified as a low pathogenic avian influenza A virus because it only caused a drop in egg production with minor other clinical signs and no mortality.

Previously, we have shown that intramuscular vaccination of chickens with the eukaryotic expression vector (EEV), expressing the influenza H5 hemagglutinin (H) protein, can stimulate a measurable and protective antibody response. Based on these results, we cloned other H genes from Eurasian H5, North American and Eurasian H7, and H15 influenza viruses into the EEV for use in vaccination of chickens to produce reference antibodies for diagnostic purposes, such as the hemagglutination inhibition (HI) test. Three-week-old specific pathogen free (SPF) chickens were vaccinated with 100 microg of EEV mixed with a cationic lipid by intramuscular injection. Then the birds were boostered twice at monthly intervals after the original vaccination. Measurable antibody titers were present for most birds after 1 month and generally increased after each boost. To examine the cross reactivity of the sera with other subtypes, HI test was conducted with antigens prepared from 15 subtypes of influenza virus. Subtype specificity of the antisera prepared by DNA vaccination were comparable or better than the antisera prepared by traditional method using whole virus vaccination. Preparation of reference antisera by DNA vaccination holds good promise because it is safe and allows for the production of H specific antibodies without producing antibodies specific to other influenza viral proteins.