Seroepidemiologic Survey of Potential Pathogens in Obligate and Facultative Scavenging Avian Species in California

Straub, Mary H.; Kelly, Terra R.; Rideout, Bruce A.; Eng, Curtis; Wynne, Janna; Braun, Josephine; Johnson, Christine K.

Blood was drawn from the metatarsal or brachial vein and aliquoted into additive-free, EDTA, and lithium heparin blood collection tubes (Becton Dickinson, Franklin Lakes, NJ). Samples were kept cool on ice for transport back to the University of California, Davis where they were aliquoted and stored at -80°C until shipment to the diagnostic laboratories. Serological testing for avian adenovirus, Chlamydophila psittaci, infectious bronchitis virus (IBV) (Arkansas (Ark), Connecticut (Conn) and Massachusetts (Mass) strains), Mycoplasma gallisepticum, Mycoplasma synoviae, avian paramyxovirus-1 (Newcastle Disease virus), avian paramyxovirus-2, avian paramyxovirus-3, and avian reovirus was performed at Texas Veterinary Medical Diagnostic Laboratory (College Station, TX) using assays optimized for poultry species. Because these assays have not been validated in condors, vultures, or eagles, we relied on cutoff titers established for poultry. Exposure to avian adenovirus was evaluated using an agar gel immunodiffusion (AGID) test. Chlamydophila psittaci exposure status was determined by a direct complement fixation (DCF) assay. Exposure status for the three strains of IBV was determined by hemagglutination inhibition (HI) assay. A titer of 1:16 or greater was considered positive for exposure to IBV. Mycoplasma gallisepticum exposure status was determined using serial tests. Samples were initially screened using a plate agglutination assay. Any samples that were positive on the first assay were then tested by HI. A titer of 1:80 or above on the HI assay was considered positive. A sample testing positive on both the plate agglutination test and HI assay was considered positive for exposure to M. gallisepticum. Mycoplasma synoviae exposure status was determined in the same manner as M. gallisepticum. Avian paramyxovirus 1, 2 and 3 exposure status was determined by HI. A titer of 1:16 or greater on the HI assay was considered positive for each of the three paramyxoviruses. Avian reovirus exposure status was determined using AGID. The number of individuals tested varied between pathogens due to limitations in sample volume. Serological testing for Toxoplasma gondii was performed at University of California, Davis (Department of Pathology, Microbiology, and Immunology, University of California-Davis) using a T. gondii agglutination test kit (Eiken Chemical Co., LTD. Tokyo, Japan, distributed by Tanabe USA, Inc., San Diego, CA). Manufacturer's recommendations were followed, and a titer of 1:32 or greater was considered positive. Testing for arboviruses common to California was performed at the Center for Vectorborne Diseases (University of California-Davis). West Nile virus titers were determined using an indirect enzyme immunoassay (EIA) as previously described [75] [76] [77] [78] . West Nile Virus cross reacts with St. Louis encephalitis virus (SLEV) and Western equine encephalitis virus on EIA, so EIA positive samples were tested using end-point plaque neutralization (PRNT) assays using the NY99 strain of WNV and the Kern 217 strain of SLEV. The PRNT assays were performed using >75 plaque forming units of virus grown on Vero cell culture. To be considered positive, sera had to neutralize >90% of the virus in at least a 1:4 dilution. Sera from free-flying California condors were also evaluated for active WNV infection by real time-polymerase chain reaction (rt-PCR) using primers as previously described [79, 80] . Additionally, free-flying condors th