AVIAN DISEASES 51:140145, 2007

Case Report
Clinical Findings, Lesions, and Viral Antigen Distribution in Great Gray Owls (Strix nebulosa) and Barred Owls (Strix varia) with Spontaneous West Nile Virus Infection
Hugo Lopes,A Pat Redig,A Amy Glaser,B Anibal Armien,C and Arno WunschmannCD
A

The Raptor Center, College of Veterinary Medicine, University of Minnesota, 1920 Fitch Avenue, St. Paul, MN 55108 B Animal Health Diagnostic Center, Cornell University, Ithaca, NY 14852-5786 C Department of Veterinary Population Medicine, College of Veterinary Medicine, University of Minnesota, 1333 Gortner Avenue, St. Paul, MN 55108 Received 21 July 2006; Accepted 3 November 2006

SUMMARY. West Nile Virus (WNV) infection manifests itself clinically and pathologically differently in various species of birds. The clinicopathologic ndings and WNV antigen tissue distribution of six great gray owls (Strix nebulosa) and two barred owls (Strix varia) with WNV infection are described in this report. Great gray owls usually live in northern Canada, whereas the phylogenetically related barred owls are native to the midwestern and eastern United States and southern Canada. Naturally acquired WNV infection caused death essentially without previous signs of disease in the six great gray owls during a mortality event. Lesions of WNV infection were dominated by hepatic and splenic necrosis, with evidence of disseminated intravascular coagulation in the great gray owls. WNV antigen was widely distributed in the organs of the great gray owls and appeared to target endothelial cells, macrophages, and hepatocytes. The barred owls represented two sporadic cases. They had neurologic disease with mental dullness that led to euthanasia. These birds had mild to moderate lymphoplasmacytic encephalitis with glial nodules and lymphoplasmacytic pectenitis. WNV antigen was sparse in barred owls and only present in a few brain neurons and renal tubular epithelial cells. The cause of the different manifestations of WNV disease in these fairly closely related owl species is uncertain. RESUMEN. Reporte de CasoHallazgos clnicos, lesiones y distribucion de antgeno viral en el carabo lapon (Strix nebulosa) y el buho barrado (Strix varia) con infeccion espontanea con virus del oeste del Nilo. La infeccion con el virus del oeste del Nilo se maniesta clnica y patologicamente de manera diferente en varias especies de aves. En este reporte se describen los hallazgos clnico-patologicos y la distribucion del antgeno viral del virus del oeste del Nilo en seis carabos lapones (Strix nebulosa) y en dos buhos barrados (Strix varia) infectados con el virus del oeste del Nilo. Los carabos lapones usualmente viven al norte de Canada, mientras los logeneticamente relacionados buhos barrados son nativos del medio oeste y Este de los Estados Unidos de Norte America y del sur de Canada. Infecciones con el virus del oeste del Nilo adquiridas naturalmente causaron la muerte sin signos previos de seis carabos lapones en un evento de mortalidad. Las lesiones predominantes de la infeccion con virus del oeste del Nilo fueron necrosis hepatica y esplenica, con evidencia de coagulacion intravascular diseminada en los carabos lapones. El antgeno del virus del oeste del Nilo estaba ampliamente distribuido en los organos de los carabos lapones haciendo blanco en celulas endoteliales, macrofagos y hepatocitos. Los buhos barrados representaron dos casos esporadicos. Estos mostraron afecciones neurologicas con obnubilacion mental que conllevo a la eutanasia. Estas aves mostraron encefalitis linfoplasmoctica de leve a moderada con nodulos gliales y pectenitis linfoplasmoctica. El antgeno del virus del oeste del Nilo era escaso en los buhos barrados y estaba presente solo en unas pocas neuronas cerebrales y celulas epiteliales del tubulo renal. Es incierta la causa de las diferentes manifestaciones de la enfermedad del virus del oeste del Nilo en estos buhos cercanamente relacionados. Key words: great gray owl, barred owl, West Nile virus Abbreviations: PCR polymerase chain reaction; WNV West Nile virus

West Nile virus (WNV), a avivirus of the Japanese encephalitis antigen complex, was introduced to North America in 1999 (2,12). Since its introduction, there have been several reports of WNVrelated morbidity and mortality in various species of birds of prey, including hawk and owl species, and there is increasing evidence that WNV infection causes different clinicopathologic disease patterns in various avian species (4,5,6,13,16,20,21). In this report, we describe the clinical presentation and necropsy ndings of six great gray owls (Strix nebulosa) that died during a WNV-associated mortality event while being kept temporarily at a Raptor Center for rehabilitation and of two sporadic cases of WNV disease in barred owls (Strix varia), that had been presented and treated at the Raptor Center of
D

Corresponding author.

the University of Minnesota College of Veterinary Medicine for neurologic disease. During the winter of 20042005, an unusually large number of great gray owls migrated from the boreal forests of Canada to Minnesota and adjoining northern frontier states, a phenomenon that is called an irruption. These irruptions occur periodically approximately every 10 to 15 years (3). Between November 2004 and April 2005, 116 great gray owls were admitted alive to the Raptor Center, almost exclusively for traumatic injuries, including internal hemorrhage, ocular trauma, and compound fractures, most of them apparently caused by collisions with moving vehicles. Depending on the severity of the injuries, the birds were treated or euthanatized. Treated birds were subjected to appropriate medical procedures ranging from supportive care to isourane anesthesia and orthopedic

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Table 1.
Case no.

Date of admission, date of death, and initial clinical signs or problems of six great gray owls (GGOW) and two barred owls (BAOW).
Species Date of admission Date of death Major clinical problems or signs

1 2 3 4 5 6 7 8
A

GGOW GGOW GGOW GGOW GGOW GGOW BAOW BAOW

1/19/05 1/20/05 3/20/05 3/23/05 4/22/05 5/10/05 8/15/03 7/31/04

9/06/05 9/08/05 9/08/05 9/05/05 9/06/05 9/06/05 8/18/03A 8/02/04A

Ocular and beak trauma, shoulder girdle fracture Ocular trauma, shoulder girdle fracture Ocular and ear trauma, poor feather condition Corneal ulcer, wrist abrasion, poor feather condition Soft tissue wound on wing, poor feather condition Ocular trauma, poor body condition and nutritional state Emaciation, depression, and head tremors progressing to recumbency Depression, recumbency and full body tremors

Euthanatized.

surgery. As part of the admission workup, approximately 1 ml of blood was drawn from the cutaneous ulnar vein with a heparinized syringe for blood count and blood chemistry. Treated birds were released into the wild as soon as possible. At the beginning of September 2005, six recovered second-year and adult great gray owls were still at the Raptor Center in an outdoor pen pending further convalescence and release (Table 1). Their injuries and fractures had been repaired and had healed uneventfully. During the routine examinations of early September, all birds appeared normal and were being athletically reconditioned. They were fed a diet composed of eviscerated mice and cut-up rats and chickens. All of the food items were maintained frozen and were thawed immediately prior to feeding. On September 5, 2005, one owl (Case No. 4) was found dead in the pen while one owl (Case No. 1) was standing on the ground and appeared depressed. The four remaining owls appeared to be alert and were acting in a healthy manner, perching high and ying around the pen. The depressed owl was moved to an indoor rehabilitation cage, and supportive therapy was initiated. The owl exhibited decreased awareness of its surroundings. Neurologic signs other than the depressed mental state were not noted. There were no apparent physical injuries. On September 6, three owls (Case Nos. 1, 5, and 6) were found dead. Blood was collected from the remaining owls (Case Nos. 2 and 3), although they did not show any clinical signs of illness, and supportive therapy was instituted. The owls were maintained in their outdoor pen to avoid possible transmission of a possibly infectious disease to other birds at the Center and were closely monitored by the staff veterinarians. On
Table 2.

September 7, both animals were perching and ying as usual, but they did not eat voluntarily. Supportive therapy was continued. Blood chemistry did not reveal any signicant changes other than a mild elevation in creatine kinase at 843 U/liter (Case No. 2) and 568 U/liter (Case No. 3). The blood count revealed leukocytosis with heterophilia and dysmorphic cells, an indication of an acute infection (Table 2). On September 8, 2005, both owls were found dead. In August 2003, a hatch-year barred owl (Case No. 7) was admitted to the Raptor Center. Barred owls are native to the midwestern and eastern United States, including Minnesota, and southern Canada. It was moderately emaciated, mildly depressed, and exhibited occasional head tremors during the admission physical examination. It was hand-fed a diet of cut-up and eviscerated mice that had been frozen and thawed. During the following days, the bird became progressively more depressed and unable to stand, with more noticeable and continuous head tremors that progressed to full body tremors. The bird was euthanatized 3 days after admission due to the clinical deterioration. In July 2004, another hatch-year barred owl (Case No. 8) was admitted to the Raptor Center. It was unable to stand, was depressed, and had pronounced head tremors. This bird was fed a high-calorie, critical-care diet (Oxbow Carnivore Care, Oxbow, 29012 Mill Road, Murdock, NE 68407, USA) by stomach tube. Despite supportive therapy, its condition did not improve, and the bird was euthanatized 2 days later with suspected WNV disease. Blood was drawn from both birds for a complete blood count (Table 2).

Blood count values of two great gray owls (GGOW) and two barred owls (BAOW) with West Nile virus infection.
GGOW 2 (case no. 2) GGOW 3 (case no. 3) Reference range A BAOW 7 (case no. 7) BAOW 8 (case no. 8) Reference rangeB

Packed cell volume Total protein (g/dl) White blood cell count (cells/mm3) HeterophilsC LymphocytesC MonocytesC EosinophilsC Basophils Cell morphology

43% 3.4 21,500 75% (16,125) 8% (1720) 8% (1720) 9% (1935) 0% 4 toxic heterophils; azurophilic granules in lymphocytes

34% 3.2 31,500 51% (16,065) 36% (11,340) 2% (630) 11% (3465) 0% 4 toxic heterophils; azurophilic granules in lymphocytes

43% 3.3

34% 3.8

36% 4.6

44% 2.54.4

11,750 21,300 43% (5080) 75% (15,975) 35% (4102) 10% (2130) 9% (1064) 15% (3195) nrD 0% nr 0% Normal Normal morphology morphology

7200 800012,000 16% (1152) 50% (4720) 47% (3383) 22% (2059) 24% (1728) 12% (1131) 12% (864) nr 1% nr Normal nr morphology

Average values from 9 rehabilitated great gray owls at the point of release to the wild. Average values from 4 rehabilitated barred owls at the point of release to the wild. C Absolute values in parentheses (cells/mm3). D nr not recorded.
B

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Fig. 1. Diffusely ochre and slightly enlarged liver in a great gray owl with WNV infection-associated hepatic necrosis. Fig. 2. Photomicrograph of marked hepatocellular necrosis in a great gray owl with WNV infection. H&E. Bar 25 lm. Fig. 3. WNV antigen-positive endothelial cells in the cerebrum of a great gray owl. Peroxidase-based polymer system; anti-WNV antibody (clone 7H2). Mayers hematoxylin counterstain. Bar 50 lm. Fig. 4. Numerous WNV antigen-positive hepatocytes and Kupffer cells in a great gray owl. Peroxidase-based polymer system; anti-WNV antibody (clone 7H2). Mayers hematoxylin counterstain. Bar 25 lm.

The neurologic signs, slightly increased total protein, and slightly reduced leukocyte count with a preponderance of lymphocytes and monocytes in one owl (Case No. 8) were interpreted as a response to a viral infection, whereas the mild leukocytosis and heterophilia of the other owl (Case No. 7) were interpreted as a general response to an acute infectious disease. Four great gray owls and both barred owls were submitted to the University of Minnesotas Veterinary Diagnostic Laboratory for post mortem investigation. The rst two great gray owls to die (Case Nos. 1 and 4) underwent necropsy at the Raptor Center. The six great gray owls underwent necropsy up to approximately 1 to 2 days after death. The necropsies of the two barred owls were performed immediately after euthanasia. The great gray owls were in a good nutritional state. All of the great gray owls had similar lesions, whereas gross lesions other than emaciation were absent in the barred owls. The livers of the great gray owls were diffusely ochre and slightly enlarged (Fig. 1). The animals had serobrinous effusion in the cardioabdominal cavity. The spleens were moderately enlarged and had multifocal white pinpoint foci. In addition, there were subepicardial petechiae and ecchymoses in two great gray owls. Samples of various organs of all animals, including brain, heart, kidney, liver, lung, eye, and spleen,

were xed in 10% neutral buffered formalin for histologic and immunohistochemical examination. The formalin-xed tissues were parafn-embedded. Sections were cut at 4 lm and stained with hematoxylin and eosin. The histologic lesions were similar among the great gray owls. There was marked hepatocellular necrosis, necrosis of splenic ellipsoids, and moderate necrosis of renal tubular epithelial cells (Fig. 2). The intestinal mucosa was multifocally necrotic. Prominent glomerular and pulmonary capillary thrombosis was present. Few microglial nodules were present in the cerebral and cerebellar gray matter. Both barred owls had a mild to moderate lymphoplasmacytic encephalitis. Both the cerebrum and the cerebellum had perivascular inltrates, and there were microglial nodules, particularly in the molecular layer of the cerebellar gray matter. In addition, both barred owls had a mild lymphoplasmacytic and histiocytic pectenitis. A peroxidase-based polymer system (Envision-HRP; DAKO, Carpinteria, CA) was used for immunohistochemical demonstration of WNV antigen in the collected organs, as previously described (20). Numerous endothelial cells in various organs, including brain, heart, kidney, liver, and lungs, were WNV antigen-positive in the great gray owls (Fig. 3). In addition, numerous splenic ellipsoids and numerous hepatocytes and Kupffer cells were WNV antigen-

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Fig. 5. Transmission electron microscopic picture of two neighboring degenerate hepatocytes of a great gray owl. A paracrystalline array of viral particles (arrowhead) is present in the intercellular space. Nuclei of hepatocytes (N), mitochondria (M), rough endoplasmic reticulum (R), and cytoplasmic membranes (small arrows) are depicted. Bar 500 nm. Inset: Magnication of viral particles, approximately 35 to 40 nm in diameter. Bar 100 nm.

positive (Fig. 4). WNV antigen was detected in numerous renal tubular epithelial cells and crypt epithelial cells in the small intestine. A few neurons (including Purkinje cells), a few cardiomyocytes, and a few proventricular epithelial cells were WNV antigen-positive. A few cerebral neurons were WNV-antigen positive in one great gray owl. In both barred owls, a few WNV antigen-positive neurons (including a few Purkinje cells) were detected in the cerebellum and brainstem. A few renal tubular epithelial cells were WNV antigenpositive in one barred owl. WNV antigen was not detected in the heart, spleen, liver, lungs, and eye of either barred owl and in the kidney of one barred owl. Fresh brain samples, including cerebrum and cerebellum, from all animals in this study were submitted as part of the routine WNV monitoring effort to the Minnesota Department of Health for WNV-specic polymerase chain reaction (PCR) analysis (10). All samples yielded a positive result. In addition, frozen tissue homogenates of four great gray owls containing heart, lung, spleen, and kidney were submitted to the Animal Health Diagnostic Center of

the Cornell University for WNV-specic PCR analysis using the probes and protocol previously described (20), with amplication by SmartCycler technology. All tested homogenates were positive for WNV nucleic acid. Pooled samples of liver, spleen, brain, intestine, lungs, and kidney of four great gray owls (Case Nos. 2, 3, 5, and 6) were submitted for isolation of avian inuenza virus and Newcastle disease virus to the virology section of the Minnesota Veterinary Diagnostic Laboratory. Filtered tissue homogenates were serially passaged in 9- to 14-day embryonated chicken eggs by allantoic sac inoculation (1,17). The embryos of the eggs inoculated with the great gray owl samples died within 3 to 5 days after the inoculation. The embryos were stunted and discolored. Occasionally, the embryos had extensive hemorrhage. However, allantoic uid at all passage levels was negative for hemagglutinating activity against turkey erythrocytes. In addition, viral particles were not detected in the allantoic uid from the third passage by contrast electron microscopy. Bacteriologic examination of heart, liver, lungs, intestine, and spleen of four great gray owls (Case Nos. 3, 4, 5, and 6) using

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standard procedures for aerobic culture and Salmonella sp. culture resulted in the isolation of numerous nonhemolytic Escherichia coli from the intestine in two great gray owls (Case Nos. 5 and 6). Staphylococcus aureus was isolated from the intestine of two other great gray owls (Case Nos. 2 and 3), and Salmonella enterica serotype Typhimurium was isolated after enrichment from the pooled organs in one animal (Case No. 6). Transmission electron microscopy was performed on formalinxed liver samples of two great gray owls (Case Nos. 2 and 3). Viral particles, approximately 35 to 40 nm in diameter and occasionally arranged in a paracrystalline array, were detected in the intercellular space and the cytoplasm of the hepatocytes of both animals (Fig. 5). DISCUSSION The peracute course of the disease in the great gray owls, essentially with sudden death as the presenting sign, contrasted with the progression of the disease seen in the phylogenetically closely related barred owls. The observed different patterns of lesions and antigen distribution appeared to correlate with the noted clinical differences. Whereas lesions were severe, acute, and widespread in the great gray owls, the lesions in the barred owls were fairly mild, chronic, and sparse. WNV antigen was abundant and widely distributed in the organs of the great gray owls and was commonly present in endothelial cells, whereas WNV antigen was sparse and limited to a few neurons of the brain and a few tubular epithelial cells in the kidney in the barred owls. Although the differences between the great gray owls and the barred owls in this study were striking, it cannot be ruled out that the described difference is the result of individual variation in disease expression rather than interspecies variation. It seems possible that the two examined barred owls may have been individuals with an exceptional manifestation that led to the presentation at the Raptor Center, and that the disease in the majority of the barred owls takes a rapidly fatal course similar to that described in the great gray owls. However, two observations support the hypothesis that there is indeed a difference between these two species regarding the WNV-associated disease manifestation. First, a recent publication describing WNV infection in various owl species also found that at least two different patterns of WNV antigen distribution exist, depending on the affected owl species (6). As observed in the great gray owls of our study, northern owls at a Canadian owl facility, including great gray owls, had abundant antigen in blood and endothelial cells that was widely distributed in numerous organs. This pattern resembles the ndings in corvid species such as crows and blue jays (19,22). In contrast, other owl species kept at the facility under similar conditions to those of the northern owl species, as well as the barred owls of our study, had only very sparse antigen in a few tissues and none in the blood vessels (6). The clinical signs observed in both barred owls, the course of the disease, the lesions, and the antigen distribution were reminiscent of the ndings describe, for example, in the vast majority of the numerous great horned owls (Bubo virginianus) with WNV infection presented to the Raptor Center in 2002 and 2003 (21). Second, only these two barred owls were diagnosed with WNV infection at the Raptor Center in 5 years of WNV presence in Minnesota (2002 through 2006), although barred owls are fairly common in Minnesota and are regularly patients at the Raptor Center. If barred owls were as susceptible to the virus and to WNV-associated morbidity and mortality as great gray owls appear to be, a higher number of barred owl patients with WNV infection would have been expected. Furthermore, no other WNV-positive barred owl was recorded by the Minnesota

Department of Health as a result of the statewide dead bird surveillance effort between 2002 and 2006 (Neitzel, pers. comm., see Acknowledgments). The route of infection of the great gray owls is uncertain but was most likely via a mosquito vector. WNV activity in mosquitoes and birds peaked in the immediate region in late August and early September (18). Alternatively, the virus may have spread within the great gray owl group by contact or via the feco-oral route, as has been shown in crows (9). Blood analysis performed on great horned owls that were housed in the adjacent pen in the weeks after the outbreak (data not shown) failed to demonstrate any indication of infectious disease in these birds. and all of the other raptors (34 birds of various species) remained healthy. Clinical differential diagnoses for the peracute disease of the great gray owls and the 100% mortality included intoxication, gout, enterotoxemia (e.g., Clostridium perfringens), septicemia (e.g., Salmonella sp.), or viral infections (e.g., avian inuenza virus, herpesvirus, adenovirus). WNV infection was considered to be unlikely on the basis of experience with WNV disease in great horned owls and various hawk species that presented to the Raptor Center from 2002 to 2005 (20,21). Even after the results of the gross and histologic examination of the great gray owl carcasses were known, WNV disease was low on the list of differential diagnoses. The disease was dominated by marked liver necrosis, which was not detected previously in other raptor species with WNV infection diagnosed at the Minnesota Veterinary Diagnostic Laboratory. Intoxication with a hepatotoxin or infection with hepatotropic (and splenotropic) viruses, such as adenovirus, polyoma virus, and herpesvirus appeared to be more likely candidates than WNV infection, although inclusion bodies were not detected. Interestingly, a case of fatal hemorrhagic fever associated with WNV infection with widespread WNV antigen in endothelial cells was recently described in a human, although the vast majority of clinically severe or fatal human WNV cases present as neuroinvasive disease with fairly sparse intraneuronal antigen presence (8,14). The blood panels obtained from two great gray owls (Case Nos. 2 and 3) were consistent with a severe acute infection. The results of the blood chemistry were within normal limits except for the mild elevation in creatine kinase, which was attributed to the stress of handling, restraint, physical examination, and blood collection. The reason that the disease may manifest differently in great gray owls than in the phylogenetically fairly closely related barred owls is uncertain. It may be speculated that great gray owls generally lack exposure to mosquito-borne diseases, such as aviviridae infections, while barred owls may have developed some degree of immunity to aviviridae (11). Great gray owls usually live in northern latitudes that allow only for short mosquito seasons that do not support the buildup of an infectious virus load within the mosquitoes. This decreases the opportunity for a successful transmission to susceptible hosts (7,15). Therefore, great gray owls may not have acquired cross-reactive immunologic protection. In contrast, barred owls live in more southern latitudes and therefore may not be immunologically na_ to aviviridae such as WNV. Hence it appears that ive phylogenetic relationships may have less predictive value than geographic associations in determining which species of owls and perhaps other birds may be susceptible to WNV.

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ACKNOWLEDGMENTS
We are grateful to Jan Shivers, Colleen Shostedt, and Laurie Reichel (Immunohistochemistry Laboratory, College of Veterinary Medicine, University of Minnesota), Susan Fuller and David Neitzel (Minnesota Department of Health), and Melissa Laverack (Animal Health Diagnostic Center, Cornell University, Ithaca, NY) for their contribution to this study.