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Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, Georgia
Department of Medical Microbiology, College of Veterinary Medicine, University of Georgia, Athens, Georgia
Southeastern Cooperative Wildlife Disease Study, Athens, Georgia
Institute of Ecology, University of Georgia, Athens, Georgia
ABSTRACT
Immunohistochemistry and virus isolation were performed on 1,057 birds. Immunohistochemistry, virus isolation, or both found 325 birds to be West Nile virus positive. Of these, 271 were positive by both methods. These results indicate that virus isolation and immunohistochemistry are approximately equal in their ability to detect West Nile virus.
TEXT
West Nile virus (WNV) is a member of the family Flaviviridae, genus Flavivirus. It is transmitted by mosquito vectors to a variety of avian hosts and incidentally to horses and humans (1). West Nile virus was first reported in the United States in 1999 in New York, where it was associated with an outbreak that killed hundreds of birds (2-4).
Since West Nile virus is a zoonotic agent and mortality in birds has usually preceded human infection and death, primary detection of virus in birds is an important part of surveillance for this virus (5, 6). Previous studies have used immunohistochemistry (IHC) and virus isolation (VI) to diagnose West Nile virus (8, 9), but there has not been a large-scale comparison of these two methods. This study compares the results of virus isolation and immunohistochemistry for 1,057 birds.
Birds were voluntarily submitted to the Southeastern Cooperative Wildlife Disease Study through state and local health departments in Georgia. Necropsies were performed on all birds in a biosafety cabinet. Liver, kidney, brain, and heart tissues were placed in 10% buffered formalin, and aseptically obtained brain and heart tissues were collected in microcentrifuge tubes containing BA-1 solution. Immunohistochemistry and virus isolation were performed as previously described (7). A "positive result" using IHC was defined as a bird that had intracellular staining in one or more tissues. "Equivocal results" for immunohistochemistry were defined as those which were impossible to judge as positive or negative.
Submitted birds represented at least 78 species (Table 1), 16 of which were positive for West Nile virus. Comparison of immunohistochemistry and virus isolation results yielded a 95% agreement rate (990/1,039). The 18 birds with an equivocal result by IHC were excluded from this total.
For immunohistochemistry, brain, heart, kidney, and liver tissues were available for most birds (97%, 97%, 87%, and 88%, respectively). In birds that were IHC positive, brain tissue was positive in 118/285 cases (41%), heart tissue was positive in 279/285 cases (98%), kidney tissue was positive in 250/267 cases (94%), and liver tissue was positive in 240/266 cases (90%).
Staining patterns on immunohistochemistry were consistent within each tissue (Fig. 1). In liver tissue, staining was confined to Kupffer cells. In kidney tissue, staining was multifocal and centered around collecting ducts. Cells that stained appeared to be a combination of macrophages, tubular epithelial cells, and cells of unknown origin. In heart tissue, staining ranged from faint and focal to overwhelming and diffuse and was most commonly seen in myofibers and infiltrating macrophages. Staining in brain tissue was usually focal and often rare. These foci consisted of a positive neuron(s) surrounded by positive glial cells. Focal or multifocal staining of Purkinje cells and mild gliosis in the cerebellum were sometimes observed.
In 311 cases that were positive by VI, most cases (68%) were positive in both brain and heart tissue. However, 23% were positive only in brain tissue and 6% were positive only in heart tissue. In one case, only a cloacal swab was positive, and in four cases, results were recorded as positive without regard to tissue.
The high agreement rate (95%) between virus isolation and immunohistochemistry indicates that the two methods are approximately equal regarding ability to detect West Nile virus. Some cases provided equivocal results by immunohistochemistry such as those with heavy background, severe autolysis, very weak staining, or staining in unusual patterns or tissues.
Virus isolation appears slightly more sensitive in that it detected 40 cases that were negative or equivocal on immunohistochemistry whereas immunohistochemistry detected only 14 cases that were negative on virus isolation. Virus isolation has the additional advantage of allowing follow-up with reverse transcription-PCR. This confirms the presence of West Nile virus specifically and allows for identification of other viruses. Our current immunohistochemical technique uses a polyclonal antibody that cross-reacts with Saint Louis encephalitis virus. Therefore, positive diagnosis of West Nile virus requires follow-up with some other method of identification or use of a monoclonal antibody. Although none of the birds in this study were found to have Saint Louis encephalitis virus, Newcastle disease virus, Highlands J virus, and Eastern equine encephalitis virus were isolated from one, two, and three birds, respectively. The final major advantage of virus isolation is that it allows for quantitative analysis of virus in tissues.
Advantages of immunohistochemistry are a faster turnaround time (typically 2 days versus 7 to 14 days for VI) and opportunity for histopathologic examination of tissues. This allows for identification of confounding factors that might have contributed to, or even caused, death. The protocol is also easily adaptable to an automated immunostainer. Immunohistochemistry also requires less-specialized equipment, and biosafety level 3 facilities are not needed. There is less risk to laboratory personnel, since live virus is not present in formalin-fixed tissues. The main disadvantage is that results may be equivocal due to autolysis, nonspecific staining, or weak staining. Our results indicate that virus isolation still works on severely autolyzed tissue while immunohistochemistry results may be equivocal.
It is important to note that each test requires different tissues for optimal diagnostic ability. For virus isolation, brain tissue was positive in 92% of positive cases while heart tissue was positive in 75% of positive cases. By IHC, brain tissue was positive in only 40% of positive cases, whereas heart tissue was IHC positive in 96% of positive cases. Since it is possible to test multiple organs simultaneously using IHC, it is probably best to base any evaluation on several tissues rather than just one or two.
While combined use of immunohistochemistry and virus isolation may slightly improve diagnostic ability, it is not practical in terms of time or economics to use both methods for screening. The decision on which method to use may depend on availability of equipment and facilities, availability and training of personnel, and personal preference. With immunohistochemistry, laboratory personnel are not exposed to live virus beyond the initial sample collection and biosafety level 3 facilities are not required. However, we have used both methods successfully and safely and do not specifically favor one over the other.
ACKNOWLEDGMENTS
This study was primarily supported by the Georgia Department of Human Resources through the Centers for Disease Control and Prevention's "Epidemiology and Laboratory Capacity for Infectious Diseases" grant program, contract 427-93-25328. Additional support was provided by the Federal Aid to Wildlife Restoration Act (50 Stat. 917) and through sponsorship from fish and wildlife agencies in Alabama, Arkansas, Florida, Georgia, Kansas, Kentucky, Louisiana, Maryland, Mississippi, Missouri, North Carolina, Oklahoma, Puerto Rico, South Carolina, Tennessee, Virginia, and West Virginia.
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