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Vet Pathol 40:14-24 (2003)
© 2003 American College of Veterinary Pathologists

Varied Pathogenicity of a Hong Kong–origin H5N1 Avian Influenza Virus in Four Passerine Species and Budgerigars

L. E. L. Perkins and D. E. Swayne

United States Department of Agriculture, Agricultural Research Service, Southeast Poultry Research Laboratory, 934 College Station Road, Athens, GA


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
This investigation assessed the ability of the zoonotic A/chicken/Hong Kong/220/97 (chicken/Hong Kong) (H5N1) highly pathogenic avian influenza virus to infect and cause disease in zebra finches (Taeniopygia guttata), house finches (Carpodacus mexicanus), house sparrows (Passer domesticus), European starlings (Sternus vulgaris), and budgerigars (Melopsittacus undulatus) after intranasal administration. Zebra finches were the most severely affected of the five species, demonstrating anorexia, depression, and 100% mortality within 5 days of inoculation. Gross lesions in this species were absent or only mild. But histologic lesions and the corresponding viral antigen were observed in multiple organs, especially in the nasal cavity, brain, pancreas, spleen, adrenal glands, and ovary. Significant morbidity and mortality also were observed in both house finches and budgerigars. Affected birds of these two species demonstrated anorexia, depression, and neurologic signs and typically were moribund or dead within 2 days of the onset of clinical signs. Gross lesions were mild or absent in house finches and budgerigars. Histologically, the brain and pancreas were the most consistently and severely affected organs in house finches. The brain was the most affected organ in budgerigars. Unlike these three species, house sparrows suffered only mild transient depression, had no mortality, and lacked gross lesions. Viral antigen and microscopic lesions were observed only in the heart and testicle of a minority of birds of this species. Starlings demonstrated neither clinical disease nor mortality and lacked gross and histologic lesions. Viral antigen was not observed in any of the collected tissues from starlings. These results indicate that there is significant variation in the pathogenicity of the chicken/Hong Kong virus for different species of birds, including species within the same order. In addition, neurotropism is a recurrent feature among birds that eventually succumb to infection.

Key words: Birds; immunohistochemistry; influenza virus; order Passeriformes; order Psittaciformes; pathogenesis; viral disease.

Highly pathogenic avian influenza (HPAI) is a fulminating and rapidly fatal systemic disease in domestic poultry that is of international significance. In 1997, an outbreak of H5N1 HPAI involving three chicken flocks occurred in Hong Kong. The virus subsequently infected and caused serious disease in 18 humans, with six fatalities.6,7 This incident of direct avian-to-human transmission substantiated the notion that avian influenza (AI) viruses could be zoonotic pathogens. Surveillance of the Hong Kong live poultry markets (LPMs) before the 1997–1998 poultry depopulation revealed that up to 20% of the chickens and 5% of the waterfowl maintained in LPMs harbored H5N1 AI viruses.22 But there were no isolations of H5N1 viruses from other birds in or in close proximity to the LPMs, including other gallinaceous species, pigeons, and caged passerine and psittacine birds, nor from feral birds in local parks or gardens.

AI viruses have been isolated from numerous wild and domestic avian species, and wild waterfowl are regarded as the primordial reservoir hosts of these viruses.23,25 In contrast, little is known about the epizootiology and pathogenicity of influenza viruses in passerine and psittacine birds. Passerine birds are common fauna in geographic areas of intensive poultry production throughout the world. Isolation of AI viruses from member species of the order Passeriformes has been infrequent, indicating that passerine birds likely do not represent a significant reservoir of AI viruses.23 But there is evidence to support the potential involvement of passerine birds in the perpetuation and transmission of AI viruses, including HPAI viruses, in areas of intense poultry production.1,9,11,14

As in the case of the passerine species, the isolation of AI viruses from psittacine birds is an uncommon event.2,21 Most influenza viruses isolated from this order of birds have been from birds being held in quarantine after importation. This was the setting for the isolation of an H9N2 influenza virus from two ring-necked parakeets that had been recently imported from Pakistan into Japan.12 The H9N2 viruses isolated from the two parakeets shared high sequence similarity of the six internal genes with the 1997 H5N1 and the 1999 H9N2 viruses that had been transmitted directly from birds to humans. The H9 and N2 surface antigens also were highly similar to those of the zoonotic H9N2 virus. This suggests that although this order of birds does not appear to play a major role in the epidemiology of influenza A viruses, the potential for involvement of psittacine birds in harboring and transmitting influenza A viruses should not be disregarded, especially in countries actively engaged in the international trade of exotic birds.

The objective of this investigation was to ascertain the susceptibility of zebra finches, house finches, house sparrows, European starlings, and budgerigars to intranasal inoculation with an H5N1 Hong Kong–origin AI virus. The virus used in this investigation was isolated from chickens involved in the initial 1997 H5N1 HPAI outbreak. The results of this investigation show that there is a significant degree of variation in the virulence of the H5N1 HPAI virus among passerine species. In addition, the H5N1 virus demonstrated a unique tissue tropism in several of the species investigated, with neurotropism being a consistent feature in species that succumbed to infection.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Virus propagation

A stock of the A/chicken/Hong Kong/220/97 (H5N1) AI virus (chicken/Hong Kong) was produced by second passage in 10 day-old embryonated chicken eggs. Allantoic fluid from inoculated eggs was collected and diluted 1:300 in brain heart infusion medium (BHI) to obtain a final inoculum titer of 106.0 mean embryo lethal dose (ELD50) per bird. The inoculum titer was chosen to parallel previous investigations of the chicken/Hong Kong virus in other avian species.17,18 A sham inoculum was made using sterile allantoic fluid diluted 1:300 in BHI. The chicken/Hong Kong virus was isolated by Drs. Les Sims and Kitman Dyrting (Agriculture and Fisheries Department, Hong Kong).

Animals

Commercially acquired young adult zebra finches (Taeniopygia guttata) and budgerigars (Melopsittacus undulatus), and wild-captured adult house finches (Carpodacus mexicanus), house sparrows (Passer domesticus), and European starlings (Sturnus vulgaris) were used in this investigation. The wild birds were acquired through the Southeastern Cooperative Wildlife Disease Study, University of Georgia. Passerines were captured by mist netting or trapping methods in Clarke and Oconee counties in Georgia. All species were maintained for a minimum of 5 days for acclimation before inoculation. Each species was housed separately in self-contained isolation units (Mark 4, Controlled Isolation Systems, San Diego, CA), ventilated under negative pressure with high efficiency particulate air (HEPA)-filtered air, and maintained under continuous lighting. Feed varied by species to some extent, but it typically consisted of appropriate commercial seed mixes, millet spray, and fresh fruit. Starlings were also provided with mealworms and canned pet food daily. Feed and water were provided ad libitum. General care was provided as required by the Institutional Animal Care and Use Committee, as outlined in the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching.8 All experiments were performed in an USDA-certified biosafety level 3 agricultural facility at the Southeast Poultry Research Laboratory.

Experimental design

Before inoculation, blood was collected from two (starlings) or four (other species) birds to ensure that the birds were serologically naive to influenza viral antigens. All the serum samples collected tested negative for anti-influenza viral antibodies with the agar gel precipitin test. For each species, birds were divided into a control group and a virus-inoculated group. The control group contained three to four birds that were inoculated through the nares with 0.05 ml of the sham-inoculum administered by a pipette (Table 1). With the exception of the starlings, of which only one control bird was sampled at 4 days postinoculation (DPI), two sham-inoculated control birds of each species were sampled at 2 and 10 DPI (zebra finches, budgerigars) or at 14 DPI (Table 1). Multiple tissues were collected by immersion in formalin for histopathologic evaluation.


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  		Table 1. Number of control birds and birds inoculated IN with the A/chicken/Hong Kong/220/97 (H5N1) AI virus, time points of sampling control and virus-inoculated birds, and total morbidity and mortality for each species.

 
The virus-inoculated group, which contained from 4 to 11 birds (Table 1), was inoculated intranasally (IN) as the controls with 0.05 ml of inoculum containing 106.0 ELD50 of the chicken/Hong Kong virus, the same inoculum titer that was used in previous investigations of this virus in other avian species.17,18 The birds were monitored daily for clinical signs and mortality. Two birds of each species were scheduled to be euthanatized and necropsied at various time points after inoculation or as the birds became moribund or died (Table 1). Birds demonstrating overt clinical disease were preferentially selected at each time point. Gross lesions were recorded. Tissues were collected from each bird by immersion in formalin for histopathologic examination. All control and virus-inoculated birds were humanely euthanatized by intravenous administration of sodium pentobarbital (100 mg/kg body weight).

Histopathology and immunohistochemistry

Tissues for histopathologic evaluation were fixed by submersion in 10% neutral buffered formalin, routinely processed, and embedded in paraffin. Sections were made at 5 µm and stained with hematoxylin and eosin. A duplicate section was immunohistochemically stained using a mouse-derived monoclonal antibody (P13C11) specific for type A influenza virus nucleoprotein (NP) antigen as the primary antibody (SEPRL, Athens, GA). The specificity of this monoclonal antibody for the NP antigen has been previously verified.16,17 Procedures for immunohistochemistry (IHC) followed those previously described, with sections of tissues from normal and influenza virus–infected chickens serving as negative and positive controls, respectively.17 Fast red was used as the substrate chromagen, and slides were counterstained with hematoxylin. Demonstration of viral antigen was based on chromagen deposition in the nucleus, which was often accompanied by chromagen deposition within the cytoplasm. Scoring of histologic lesions and distribution of viral antigen as demonstrated by IHC were based on previously used scoring systems.17,24


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Sham-inoculated controls

Sham-inoculated control birds of each of the species used in this investigation lacked gross lesions, although histologic lesions were observed in several control birds of each species. These histologic lesions were most pronounced in the sham-inoculated controls of wild-captured birds. In the zebra finches group, one of the four control birds had moderate heterophilic bursitis. A separate single zebra finch had mild lymphoplasmacytic ingluvitis. In the house finches group, moderate multifocal lymphoplasmacytic air sacculitis was observed in one of the four control birds, and two of the control birds had mild periportal to bridging lymphohistiocytic infiltrates in the liver. In addition, nasal nematodiasis was observed in two of the four house finches used as controls; inflammation was not affiliated with the presence of nematodes in the anterior nasal cavity. Three of the four control sparrows had a mild to moderate burden of coccidia in the small intestine. Nasal nematodiasis also was observed in three out of the four sham-inoculated sparrows, and mild rhinitis with catarrhal to heterophilic exudation was affiliated with the nematodes. Of the five species investigated, lesions were most pronounced in the three starlings that served as sham-inoculated controls. These lesions included eosinophilic to lymphoplasmacytic ingluvitis with intralumenal nematodes (3/3), mild lymphoplasmacytic laryngitis and tracheitis (1/3), and moderate lymphoplasmacytic air sacculitis and polyserositis (2/3). The latter two control starlings also had nodular lymphoplasmacytic to lymphohistiocytic infiltrates in the liver. Organisms were not identified in association with these lesions in acid fast and Giemsa-stained sections of the liver. In the budgerigars group, mild lymphoplasmacytic infiltrates were observed in the submucosa of the larynx of one of the four control birds.

Infrequent nonspecific chromagen deposition was observed in secondary lymphoid tissues and rare individual submucosal cells of the respiratory, reproductive, and enteric tracts of each species. This chromagen deposition was restricted to cytoplasmic granules of scattered individual cells. Immunohistochemical staining of this nature has been interpreted previously as staining of mast cell granules.10,17

Morbidity and mortality

Morbidity and mortality were varied among the five species investigated and are summarized in Table 1. The zebra finches were the most rapidly affected of the five species investigated. For the first nine zebra finches inoculated with the chicken/Hong Kong virus, mortality began without premonitory signs of disease at 3 DPI and continued until the last bird died at 5 DPI. Moderate to severe depression was observed in all the six birds that died at 4 and 5 DPI. In addition, one of the birds that died at 4 DPI demonstrated mild neurologic signs, which were first observed at 3 DPI. Except for the two zebra finches sampled at 2 DPI, there was 100% mortality in this species (Table 1).

In the initial 11 house finches, clinical signs were not observed until 4 DPI, with mild depression in a single bird that was sampled at 4 DPI. By 5 DPI, two of the nine house finches demonstrated moderate depression, ruffled feathers, and neurologic signs that included incoordination and tremors. One bird died at 6 DPI, and the remaining birds demonstrated mild (3/5) to severe depression (2/5) accompanied by variable neurologic signs. The two most severely affected house finches were euthanatized and sampled at 7 DPI. The remaining three house finches continued to be mildly depressed until 13 DPI, at which time one of the finches was found dead. A second house finch was moribund with severe neurologic signs at 13 DPI and was therefore euthanatized. The single house finch remaining at 14 DPI was clinically normal.

In the budgerigars, moderate depression was first recognized in four of the six birds remaining at 5 DPI. Mortality began a few hours after clinical signs were first noted, and five of the six birds became moribund or died between 5 and 6 DPI. Each of these budgerigars demonstrated moderate to severe neurologic signs. The sole remaining budgerigar began to demonstrate depression and incoordination at 7 DPI. The neurologic signs in this single bird gradually progressed to pronounced opisthotonus and torticollis at 9 DPI, when the bird was euthanatized.

In contrast with the finches and budgerigars, only three of the seven virus-inoculated sparrows demonstrated transient clinical disease, which was observed between 4 and 7 DPI. The affected sparrows were moderately depressed and anorexic, and were huddled at the bottom of the cage with ruffled feathers. Two of the affected birds were euthanatized for sampling at 7 DPI. The remaining affected sparrows resumed normal feeding and activity by 8 DPI. The starlings were unique among the species investigated in that none of the four virus-inoculated starlings demonstrated any clinical aberrations during the course of the investigation.

Gross lesions

The zebra finches that died consistently had gross lesions of carcass dehydration and an absence of seed in the proximal enteric tract. Three of the birds found dead were too autolyzed for accurate assessment of gross lesions in the remaining organs. Splenomegaly was observed in three out of six of the zebra finches examined grossly. Accumulation of yellow mucinous feces in the distal intestine and cloaca was often observed in the zebra finches that died between 3 and 5 DPI (5/7).

In the house finches, dehydration and a lack of seed in the proximal enteric tract were typical observations in birds that had demonstrated neurologic signs before death. Nine of the 11 birds had splenomegaly with variable mottling of the splenic parenchyma. Five of the 11 house finches, more specifically those that were sampled between 6 and 14 DPI, had mild to pronounced mottling and firmness of the pancreas. Vent pasting with bile-tinged urates and feces was observed in the two house finches sampled at 7 DPI. Also, mild edematous thickening of the conjunctiva was observed in two birds. This lesion was associated histologically with moderate reactive lymphoid hyperplasia and was interpreted to be unrelated to inoculation with the chicken/Hong Kong virus.

Like two species of finches, the budgerigars with neurologic signs had carcass dehydration and a lack of seed in the proximal alimentary tract (6/10). Feces contained within the cloaca were watery and contained increased urates. Of the 10 budgerigars, three had vent pasting. Unlike the budgerigars and both species of finches, the seven sparrows and four starlings inoculated with the chicken/Hong Kong virus had no gross lesions.

Histopathology and immunohistochemistry

Histologic lesions and the corresponding viral antigen were distributed among multiple tissues in the zebra finches, house finches, and budgerigars. In contrast, tissues from the sparrows and starlings contained minimal or no viral antigen or lesions that could be attributed to inoculation with the chicken/Hong Kong virus. The distribution and average severity of histologic lesions and the average distribution and frequency of viral antigen are summarized in Table 2.


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  		Table 2. Distribution of histological lesions and viral antigen resultant from IN inoculation of zebra finches, house finches, sparrows, and budgerigars with the A/chicken/Hong Kong/220/97 (H5N1) AI virus.

 
Zebra finches. Of the five species investigated, the most widespread distribution of histologic lesions and viral antigen was observed in the zebra finches (Table 2). Although the virus demonstrated particular tropism for the nasal cavity, brain, adrenal gland, pancreas, spleen, and ovary in this species, almost every tissue examined histologically contained viral antigen. Mild to severe necrotizing rhinitis and sinusitis were observed in zebra finches that were sampled or died between 2 and 4 DPI (5/6). Viral antigen was present in epithelial cells, submucosal and intraepithelial leukocytes, and infrequent vascular endothelial cells. In the brain, viral antigen was present in neurons, ependymal cells, epithelial cells of the choroid plexus and glia, and endothelial cells (4/5). But lesions in the brain were minimal to mild and included perivascular edema and infrequent foci of a few necrotic cells that related to the presence of viral antigen (3/5). Likewise, in the adrenal gland, viral antigen was more widely distributed than were histologic lesions. Lesions consisted of minimal to mild vacuolar degeneration to necrosis of corticotropic and chromaffin cells containing viral antigen (Fig. 1a). Only the zebra finches sampled at 4 and 5 DPI had lesions and the corresponding viral antigen in the adrenal glands (2/4) (Fig. 1b), although sections of this organ were not obtained from birds collected at 3 DPI. Of the tissues examined histologically, the pancreas was the most severely affected, with wide distribution of viral antigen. Moderate to severe multifocal to confluent pancreatic acinar necrosis occurred consistently among the zebra finches euthanatized or dying between 2 and 5 DPI (5/6). Viral antigen (5/6) corresponded to the acinar necrosis and was distributed diffusely in the pancreatic acinar epithelium of birds sampled between 3 and 5 DPI. In the spleen, sinusoidal histiocytosis, histiocytic necrosis, lymphoid depletion, and sinusoidal congestion were the typical lesions (5/6) (Fig. 2a). Between 2 and 5 DPI, viral antigen was predominantly localized to splenic histiocytes, smooth muscle cells of arteriolar walls, and endothelial cells (6/6) (Fig. 2b). In contrast to the testicle, in which there were neither viral antigen nor histologic lesions, the ovary of two out of three females did not demonstrate viral antigen and necrosis. Viral antigen was localized within thecal epithelial cells and medullary cells but was more consistently present in the former. Other cell types in which viral antigen was demonstrated in the zebra finches included the ciliated epithelial cells and leukocytes of the air sac (3/6), pulmonary endothelial cells (2/6), cardiac myocytes (4/6), neurons in peripheral ganglia (1/6), enteric epithelial cells (4/6), ventricular smooth muscle cells (3/6), hepatic endothelial cells, Kupffer cells, and hepatocytes (4/6), renal tubular epithelial cells and glomerular endothelial cells (3/6), skeletal myofibers (1/6), and follicular epithelial cells of the skin (1/6). Viral antigen was infrequently to rarely observed in most of these tissues (Table 2). Minimal to mild parenchymal necrosis that was inconsistently accompanied by heterophilic to mixed inflammatory infiltrates was variably associated with the presence of viral antigen in these tissues (Table 2). Neither lesions nor viral antigen were observed in the bone marrow, Harderian or lacrimal glands, or thyroid or parathyroid glands, the latter of which were only sporadically included in the tissues sampled.



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  		Fig. 1. Adrenal gland: Zebra finch, 4 DPI. Fig. 1a. Focal vacuolar degeneration to necrosis of adrenal corticotrophic cells. HE. Bar = 50 µm. Fig. 1b. Chicken/Hong Kong viral antigen shows a diffuse distribution in parenchyma of the adrenal gland, localizing in both corticotrophic and chromaffin cells. Biotin-streptavidin complex with hematoxylin counterstain. Bar = 50 µm.
Fig. 2. Spleen; zebra finch, 4 DPI. Fig. 2a. Sinusoidal histiocytosis with histiocytic necrosis. HE. Bar = 50 µm. Fig. 2b. Splenic histiocytes, vascular endothelial cells, and myocytes of the arteriolar tunica media containing chicken/Hong Kong viral antigen. Biotin-streptavidin complex with hematoxylin counterstain. Bar = 50 µm.
Fig. 3. Pancreas; house finch, 4 DPI. Fig. 3a. Severe diffuse necrotizing and suppurative pancreatitis. HE. Bar = 100 µm. Fig. 3b. Chicken/Hong Kong viral antigen distributed among remanant islands of pancreatic acinar epithelium. Biotin-streptavidin complex with hematoxylin counterstain. Bar = 100 µm.

 
House finches. The distribution of histologic lesions and viral antigen in the house finches paralleled that observed in the zebra finches, although generally lesions were less prominent and viral antigen was less frequently present within the affected tissues. The heart, brain, adrenal glands, pancreas, spleen, and testicle were the tissues that were most affected in this species. As in the case of the zebra finches, the pancreas was the most consistently and severely affected of these six tissues. Pancreatic necrosis with heterophilic to mononuclear inflammation (6/10) was routinely observed in house finches euthanatized or dying at 4, 7, and 13 DPI (Fig. 3a). There was patchy distribution of viral antigen in the pancreatic acinar epithelium of the affected birds (6/10) (Fig. 3b). In the heart, scattered foci of myofiber hyalinization and fragmentation with mild mononuclear infiltrates were directly associated with the presence of viral antigen (6/11). In the brain, necrosis (6/11) and the corresponding viral antigen (6/11) were demonstrated in randomly scattered foci of neurons, glial cells, and ependymal cells (Fig. 4a, b). Mild multifocal acute adrenal necrosis, which again was affiliated with the presence of viral antigen, was observed in five out of nine house finches. Histologic lesions and viral antigen were absent in each of these tissues in house finches sampled at 2 and 14 DPI. But the exception to this was the spleen, in which viral antigen (6/11) was demonstrated in the nucleus and cytoplasm of leukocytes at 2 DPI as well as at 4, 7, and 13 DPI. Splenic lesions were variable and included congestion, individual cell necrosis, sinusoidal histiocytosis, and mild hemosiderosis (6/11). Of the six male house finches from which tissues were collected, two had moderate testicular degeneration at 7 DPI, which corresponded to the presence of viral antigen in the Sertoli cells of the seminiferous tubules.



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  		Fig. 4. Cerebellum; house finch, 7 DPI. Fig. 4a. Focal neuronal necrosis involving all three cerebellar layers and associated rarefaction of the neuropil. HE. Bar = 100 µm. Fig. 4b. Chicken/Hong Kong viral antigen demonstrated in neurons of all three cerebellar layers. Biotin-streptavidin complex with hematoxylin counterstain. Bar = 100 µm.
Fig. 5. Heart; sparrow, 7 DPI. Fig. 5a. Focal lymphohistiocytic myocarditis. HE. Bar = 100 µm. Fig. 5b. Chicken/Hong Kong viral antigen in few degenerate cardiac myocytes and affiliated histiocytes. Biotin-streptavidin complex with hematoxylin counterstain. Bar = 100 µm.
Fig. 6. Testicle; sparrow, 7 DPI. Fig. 6a. Degeneration to necrosis of Sertoli cells with early interstitial mononuclear infiltrates. HE. Bar = 100 µm. Fig. 6b. Intranuclear and cytoplasmic localization of chicken/Hong Kong viral antigen in Sertoli cells. Biotin-streptavidin complex with hematoxylin counterstain. Bar = 100 µm.

 
Several other tissues in the house finches contained viral antigen rarely to infrequently, though histologic lesions associated with the presence of viral antigen were minimal or absent. In the nasal cavity, four house finches had moderate to severe lymphoplasmacytic to pyogranulomatous rhinitis and sinusitis. Of these four finches, two had nematodes associated with rhinitis; the lesions observed in these four were thus interpreted to be unrelated to viral inoculation. But viral antigen was observed in scattered epithelial cells and submucosal leukocytes of 7 of the 11 birds sampled between 2 and 13 DPI. Mild necrosis to heterophilic inflammation was observed in association with viral antigen in the nasal cavity (7/11). Other organs in which viral antigen and minimal to mild necrosis were observed in this species included the ovary (1/5), peripheral ganglia (3/11), Harderian and/or lacrimal glands (3/11), and feather follicles (2/11). The liver of 10 of the 11 birds contained mononuclear infiltrates centered around the portal triads. This lesion again was interpreted to be unrelated to viral infection. Viral antigen was observed in the liver in rare Kupffer cells of finches sampled at 7 and 13 DPI. Similarly, viral antigen was demonstrated rarely to infrequently in parabronchiolar epithelial cells and interstitial leukocytes (4/10), renal tubular epithelial cells (2/11), and skeletal myofibers (1/11). Histologic lesions were not observed in direct association with viral antigen in the liver, lung, kidney, or skeletal muscle of this species. Viral antigen was not detected in the conjunctiva, larynx, or air sac, although mild to moderate reactive lymphoid hyperplasia was observed in these tissues in one, two, and three birds, respectively. These lesions were interpreted as incidental lesions unrelated to viral inoculation. Lesions and viral antigen were absent in the trachea, alimentary tract, bone marrow, and thyroid and parathyroid glands of the house finches.

Budgerigars. Compared with the two species of finches, the distribution of histologic lesions and viral antigen was more restricted in budgerigars (Table 2). The brain was distinct among the tissues examined histologically. As in the case of the two species of finches, viral antigen was randomly distributed in the brain of the budgerigars among neurons, glial cells, ependymal cells, and the epithelium of the choroid plexus (5/8). Histologic lesions were coupled to the presence of viral antigen and included mainly perivascular edema and necrosis of cells containing viral antigen (4/8), although minimal heterophilic inflammation was observed in the stroma of the choroid plexus of one budgerigar. The nasal cavity (2/8), air sac (1/7), proventriculus (3/8), kidney (2/7), spleen (4/8), ovary (1/2), and parathyroid gland (1/1) also contained viral antigen, although the presence of viral antigen in these tissues was infrequent to rare. Necrosis and minimal to mild heterophilic inflammation were in direct correlation with the presence of viral antigen in each of these tissues. In addition, mild sinusoidal histiocytosis with increased erythrophagocytosis was observed in the spleen. Lesions and viral antigen were not observed in the larynx, trachea, lung, adrenal gland, heart, pancreas, testicle, skeletal muscle, integument, bone marrow, thymus, bursa, and Harderian or lacrimal glands of the budgerigars.

Sparrows. Histologic lesions that were directly affiliated with the presence of viral antigen in sparrows were confined to the heart and the testicle. Mild multifocal lymphohistiocytic infiltration was observed in the heart of two sparrows at 7 and 14 DPI (2/6) (Fig. 5a), whereas viral antigen was demonstrated in a few degenerative cardiac myocytes and histiocytes of both sparrows sampled at 7 DPI (2/6) (Fig. 5b). The same sparrows with myocarditis at 7 and 14 DPI also had severe testicular lesions that varied from severe degeneration to necrosis of Sertoli cells at 7 DPI to severe tubular atrophy with multifocal to confluent interstitial lymphohistiocytic infiltrates at 14 DPI (2/3) (Fig. 6a). Near-diffuse localization of viral antigen was demonstrated in Sertoli cells at 7 DPI, but viral antigen was not present in the testicle at 14 DPI (Fig. 6b).

Other lesions in the sparrows included mild reactive lymphoid hyperplasia in the conjunctiva (2/6) and mild intestinal coccidiosis (1/6). These lesions were interpreted to be incidental findings unrelated to inoculation with the chicken/Hong Kong virus. All other tissues examined histologically lacked lesions and viral antigen.

Starlings. Viral antigen was not demonstrated in any of the tissues examined in starlings. There were, however, multiple lesions in this species that were interpreted to be unrelated to inoculation with the chicken/Hong Kong virus. These lesions included mild eosinophilic rhinitis with intralesional nematodes (1/4), mild lymphoplasmacytic laryngitis (2/4), moderate lymphoplasmacytic air sacculitis (3/4), mild pneumoconiosis (1/4), mild lymphohistiocytic interstitial pneumonia with intravascular microfilaria (2/4), mild to moderate eosinophilic to lymphoplasmacytic ingluvitis with intralumenal nematodes (4/4), moderate multifocal lymphoplasmacytic to granulomatous hepatitis (4/4), and moderate lymphoplasmacytic polyserositis (2/4). A few nematodes also were observed within the lumen of the distal small intestine in two birds. Other organs lacked histologic lesions.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Intranasal administration of the chicken/Hong Kong virus resulted in high morbidity and mortality in zebra finches, house finches, and budgerigars within 10 days of IN inoculation. Clinically, the affected birds suffered a sudden onset of mortality, severe depression, and/or neurologic dysfunction. Clinical results of inoculation of these nongallinaceous species with the chicken/Hong Kong virus were comparable with those previously reported for this and other HPAI viruses in chickens and turkeys, indicating that the chicken/Hong Kong virus is highly pathogenic for these avian species as well.17,25 Furthermore, using immunohistochemistry, viral antigen was demonstrated in multiple tissues, indicating that disseminated infection, again typical of HPAI viruses in domestic poultry, occurred in finches and budgerigars. This is the first investigation to detail the clinical disease, gross and histologic lesions, and distribution of viral antigen after infection of passerine and psittacine species with an H5N1 HPAI virus.

In contrast to finches and budgerigars, inoculation of sparrows and starlings with the chicken/Hong Kong virus did not result in mortality, and only transient morbidity was observed in a few of the sparrows. In addition, viral antigen was demonstrated in only a small number of tissues from a minority of the sparrows and was not demonstrated in tissues from the starlings. Interestingly, those sparrows in which viral antigen and/or histologic lesions were demonstrated were males, suggesting that male sparrows may have enhanced susceptibility to infection with this particular influenza viruses. These results contrast with previous experimental inoculation of starlings and sparrows with the A/chicken/Victoria/1/85 (H7N7) and the related A/Starling/Victoria/5156/85 (H7N7) HPAI viruses, both of which caused 100% mortality in starlings and 30% mortality in sparrows.14

In summary, the chicken/Hong Kong influenza virus demonstrated distinctive grades of virulence among the four passerine species used in this investigation and covered a spectrum of virulence analogous to the general spectrum of pathogenicity of AI viruses in domestic poultry.3 Furthermore, this investigation demonstrates that a single influenza A virus may demonstrate substantial variation in virulence among different avian species, including species within the same order.17,18

Through the application of immunohistochemistry, the localization of viral antigen was found to closely correlate to the clinical manifestations of disease in each species investigated. The demonstration of the chicken/Hong Kong viral antigen in the brain was most intriguing. Antigen-containing neurons and glia in the central nervous system were repeatedly observed in the two species of finches and the budgerigars that had died or became moribund after IN administration of the chicken/Hong Kong virus. In addition, viral antigen was present in peripheral ganglia in both species of finches. Conversely, viral antigen was not demonstrated in the brain or peripheral nervous tissues of either sparrows or starlings, both of which survived with mild or no morbidity, respectively. Combined with the results of previous investigations of the chicken/Hong Kong virus in other species of birds, these results indicate that the immune-privileged nervous system is a preferred site of replication of the chicken/Hong Kong in susceptible avian species.17,18 Furthermore, neurotropism is likely to be the predominant factor in the production of morbidity and mortality relative to infection with this HPAI virus in nongallinaceous species.

Apart from the brain, there was a commonality among the susceptible species used in this investigation in the localization of viral antigen in several other tissues, including the heart, pancreas, spleen, nasal epithelium, and reproductive organs. Previous investigations also have shown that these tissues are common sites for the localization of the chicken/Hong Kong virus in other avian species.17,18 In addition, other subtype H5 and H7 HPAI viruses have shown a similar predilection for localization and replication within these tissues.4,5,13,24 In a diagnostic sense, these tissues in particular should be considered as the optimal sites for routine sampling in suspected cases of HPAI in both gallinaceous and nongallinaceous species.

The avian order Passeriformes is one of the most diverse orders of birds, with member species occupying a wide variety of environmental niches throughout the world. Although the geographic distribution of the order Psittaciformes is more limited, certain psittacine species have become widely distributed because of exotic bird trades, captive breeding, and establishment of isolated feral populations. The role of both passerine and psittacine birds in the natural epidemiology of AI is considered to be only minor. But previous reports provide evidence that both passerine and psittacine birds can harbor AI viruses, including HPAI viruses.2,9,11,12,14,15,19–21 The current investigation also supports this conclusion by demonstrating the susceptibility of several passerine and a psittacine species to IN inoculation with a zoonotic H5N1 HPAI virus. Despite the lack of isolation of H5N1 viruses from birds in LPMs and from feral birds in parks and gardens in Hong Kong in 1997, the results of this investigation suggest that passerine and psittacine birds may participate in the perpetuation of H5N1 and possibly other AI viruses isolated from birds in LPMs in Hong Kong.22 But whether these nongallinaceous species can serve as biologic vectors or are simply dead-end hosts requires further investigation. Furthermore, considering the variation of virulence that the chicken/Hong Kong virus demonstrated among the passerine species in this investigation, it is difficult to predict the susceptibility of other passerine species to this or other HPAI viruses. In a global sense it is important to raise the awareness of the possible impact that active intra- and international trade of passerine and psittacine birds could have in heightening the transmission of AI viruses among avian species.


   Acknowledgments
 
We extend special thanks to the Southeastern Cooperative Wildlife Disease Study for providing the wild birds used in this investigation. Special thanks also go to Joan Beck, Elizabeth Turpin, and Roger Brock for their technical assistance.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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Request reprints from Dr. David E. Swayne, United States Department of Agriculture, Agricultural Research Service, Southeast Poultry Research Laboratory, 934 College Station Road, Athens, GA 30605 (USA). E-mail: dswayne{at}seprl.usda.gov.




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