West Nile virus outbreak in North American Owls, Ontario, 2002

Emerging Infectious Diseases, Dec, 2004 by Ady Y. Gancz, Ian K. Barker, Robbin Lindsay, Antonia Dibernardo, Katherine McKeever, Bruce Hunter

Sample Collection

Complete diagnostic necropsies were performed at the Ontario Veterinary College on 94 owls and one falcon (family Falconidae) that died at the Owl Foundation from April 15 to December 25, 2002. This broad time frame was to facilitate detection of the first and last WNV-related deaths in the 2002 outbreak. All carcasses were kept frozen at -20[degrees]C from shortly after death and were allowed to thaw at 4[degrees]C for 24 to 48 h before examination (March-August 2003). For each bird, samples of brain, lung, liver, spleen, and kidney were pooled and refrozen at -70[degrees]C until analyzed by real time RT-PCR. Small core samples of brain, kidney, and liver were collected with a 20-gauge spinal needle from three additional carcasses that had not been available for necropsy.

During the outbreak, several hundred louse flies were collected at the foundation and frozen at -20[degrees]C. These flies were collected off sick or dead birds, as well as from birds that appeared healthy. Six flies were submitted for species identification, while whole-body homogenates of 23 flies, including 18 that were removed from dead or sick owls, were tested for WNV by real time RT-PCR.

From January 22 to May 1, 2003, a serologic survey of all outbreak survivors was conducted. Blood samples were collected from the jugular or cutaneus ulnar vein and placed in heparinized tubes. Plasma was then separated by centrifugation and frozen at -70[degrees]C until analyzed.

Real-time RT-PCR

Real-time RT-PCR assay was used to detect WNV RNA as previously described (15). Samples consisted of homogenates prepared from pooled brain, lung, liver, spleen, and kidney (approximately 1 [mm.sup.3] of each tissue). RNA was extracted by using the RNeasy 96 viral isolation kit (Qiagen, Inc.,Valencia, CA). The ABI Prism 7700 Sequence Detection System and the TaqMan One Step PCR Master Reagents Kit (PE Applied Biosystems, Foster City, CA) were used for the assay. Positive controls consisted of three 10-fold dilutions of Egypt 101 (Eg 101) strain of WNV. Three sets of negative (water) controls were used, two during the extraction procedure and one during amplification (i.e., no template). Extracts were screened with the generic 3' NC primer set. Positive samples underwent a second RNA extraction and were then tested with both the 3' NC and WNV primer sets. Primer sequences were as described (15). Samples that had threshold cycle [less than or equal to] 37 with both primer sets were considered positive. Homogenates prepared from whole louse flies were tested with the same procedures described for owl tissues.

Serologic Testing

We used an enzyme-linked immunosorbent assay (ELISA) recently shown to detect avian anti-WNV immunoglobulin (Ig) G in 23 avian species of 12 orders, including a Barred Owl (Strix varia) (6). The assay was performed as described, with slight modifications. Briefly, the inner 60 wells of a 96-well plate were coated with the monoclonal antiflavivirus antibody 4G2. After the wells were treated with blocking butler, WNV recombinant COS-1 viral antigen (Centers for Disease Control and Prevention, Atlanta, GA) and a control recombinant COS-1 antigen (Centers for Disease Control and Prevention) diluted to 1:100 were added to the top and bottom 48 wells of the plate, respectively. Plasma samples diluted 1:400 were added, as were positive and negative controls. Horseradish peroxidase-conjugated goat anti-wild bird IgG (Bethyl Laboratories, Inc., Montgomery, TX) was added and allowed to bind to anti-WNV antibodies before the substrate tetramethylbenzidine (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD) was added. The reaction was stopped after 30 min by [H.sub.2]S[O.sub.4] and read by a microtiter plate reader at 450 nm. Samples showing positive and negative optical density ratios >2 at the 1:400 dilution were considered positive.

A subset of 20 plasma samples representing eight species of owls and four species of raptors was tested by plaque reduction neutralization tests (PRNT). The assay was performed as described (4). Titers were expressed as the highest dilution that produced >90% reduction in plaque number. Titers [greater than or equal to] 40 were considered positive. The controls used were back titrations of WNV (NY strain) at 2.5 x [10.sup.7] PFU/mL diluted to 100, 10, and 1 PFU and a negative control (media only). PRNT assays were performed in a biosafety level 3 facility at the National Microbiology Laboratory, Winnipeg, Manitoba.