Highly Pathogenic Avian Influenza Virus (H5N1) Infection in Red Foxes Fed Infected Bird Carcasses

Leslie A. Reperant; Geert van Amerongen; Marco W.G. van de Bildt; Guus F. Rimmelzwaan; Andrew P. Dobson; Albert D.M.E. Osterhaus; Thijs Kuiken

Disclosures

Emerging Infectious Diseases. 2008;14(12):1835-1841. 

In This Article

Results

Clinical Signs

Clinical signs were not observed in foxes infected intratracheally or in foxes fed infected bird carcasses. However, body temperature of 2 of the 3 foxes infected intratracheally (nos. 1 and 2) and of 1 of the 3 foxes fed infected bird carcasses (no. 5) rose from 38.5°C-39°C (reference range) to 40°C-40.5°C at 2 to 4 dpi. No clinical signs and no rise in body temperature were observed for the negative-control foxes (nos. 4 and 8).

Virology

The virus was isolated from pharyngeal swabs from all infected foxes and from nasal and rectal swabs from 1 fox and rectal swabs from another (Figure 1). Foxes infected intratracheally excreted the virus through the pharynx from 1 dpi on, up to 3-7 dpi; peak titers of pharyngeal excretion were 103.5-105.2 TCID50/mL at 1-3 dpi. Foxes that had been fed infected bird carcasses excreted the virus through the pharynx from 1 dpi on, up to 3-5 dpi; peak titers of pharyngeal excretion were 104.2-104.5 TCID50/mL at 1 dpi. Student t test showed no significant difference in the patterns of pharyngeal excretion between the 2 groups of foxes according to areas under the curve (t = -0.667, df = 4, p = 0.54). No virus was isolated from any swabs from any negative-control foxes.

Figure 1.

Infectious virus titers obtained from pharyngeal, nasal, and rectal swabs of foxes infected intratracheally with highly pathogenic avian influenza (HPAI) virus (H5N1) (left, black symbols) or fed chicks infected with HPAI virus (H5N1) (right, gray symbols) at various time points after infection. No virus was isolated from any swabs of the negative-control foxes. TCID50, median tissue culture infectious dose.

The virus was isolated from the trachea (102.6 TCID50/g tissue) and lung (103.3 TCID50/g) of 1 of 3 foxes infected intratracheally (no. 2), and from the tonsil (102 TCID50/g) of another fox infected intratracheally (no. 3). No virus was isolated from any of the organs of the foxes fed infected bird carcasses or of the negative-control foxes.

Gross Examination

Of the 3 foxes infected intratracheally, 2 (nos. 1 and 2) had extensive multifocal or coalescing pulmonary lesions, which were dark purple and slightly firm (Figure 2). The estimated percentage of affected lung tissue was 20% (no. 1) and 80% (no. 2). In contrast, 1 of the 3 foxes infected intratracheally (no. 3) and all foxes fed infected bird carcasses had ≥2 small multifocal lesions (1-5 mm), which affected <5% of the lungs. In addition, 2 of the 3 foxes fed infected bird carcasses (nos. 5 and 6) had randomly distributed petechial hemorrhages throughout the lungs. Moderate enlargement of the spleen, tonsils, and/or tracheobronchial lymph nodes was observed in all foxes, whether infected intratracheally or fed infected bird carcasses. Negative-control foxes had no respiratory or extrarespiratory lesions.

Figure 2.

Lesions and associated expression of influenza virus antigen in respiratory and extrarespiratory organs of foxes infected intratracheally with HPAI virus (H5N1), at 7 days postinoculation. A) Lungs of control fox sham-inoculated with phosphate-buffered saline. B) Lungs of intratracheally inoculated fox presenting extensive consolidated lesions (darkened areas), characterized by C) diffuse alveolar damage and regeneration (type II pneumocyte hyperplasia) and D) expression of infl uenza virus antigen in the nucleus and, to a lesser extent, cytoplasm of mononuclear and epithelial cells. E) Focus of infl ammation and cardiomyocytic necrosis in the heart, associated with F) expression of infl uenza virus antigen in the nucleus of cardiomyocytes. G) Focus of gliosis and neuronal necrosis in the cerebrum, associated with H) expression of infl uenza virus antigen in the nucleus and, to a lesser extent, cytoplasm of glial cells and neurons. Panels C-H, original magnifi cation ×40.

Histopathologic Findings

Histologic lesions were found in foxes infected intratracheally and in foxes fed infected bird carcasses. However, the lesions were more severe in foxes infected intratracheally

Table

The 2 most severely affected foxes (nos. 1 and 2, infected intratracheally) had severe hemorrhagic bronchointerstitial pneumonia with extensive coalescing lesions of inflammation and necrosis, characterized by macrophage and neutrophil infiltration of the alveolar walls and loss of histologic architecture. The alveolar and bronchiolar lumina were filled with alveolar macrophages, neutrophils, and erythrocytes, mixed with fibrin and cellular debris. In both foxes, sloughing of the alveolar and bronchiolar epithelia indicated necrosis, and type II pneumocyte and bronchiolar epithelial hyperplasia indicated regeneration (Figure 2). The other foxes (no. 3, infected intratracheally, and all foxes fed infected bird carcasses) had minimal to mild bronchointerstitial or interstitial pneumonia. They had small- to medium-sized foci of inflammation in the lungs, located mostly around the bronchioles and characterized by thickened alveolar walls that were infiltrated with macrophages and neutrophils. Type II pneumocyte and bronchiolar epithelial hyperplasia was observed in the lungs of fox no. 7. Respiratory organs of negative-control foxes had no lesions.

Extrarespiratory histologic lesions were seen only in foxes infected intratracheally, namely, in the heart of fox no. 2 and in the cerebrum of foxes nos. 1 and 2. Fox no. 2 had multiple inflammatory and necrotic lesions in the myocardium, characterized by infiltration of macrophages and neutrophils and necrotic cardiomyocytes (Figure 2). The cerebrum of foxes nos. 1 and 2, infected intratracheally, had multiple lesions of acute to subacute encephalitis, from mild to severe, characterized by perivascular cuffing, foci of gliosis or neuronal necrosis, or a combination of these lesions; their cerebellum and brain stem did not show any lesions (Figure 2). No relevant lesions were seen in other organs, including organs of the digestive tract, of any other foxes.

Immunohistochemical Findings

Cells expressing the influenza virus antigen were present in the lungs, heart, and brain of 1 of 3 foxes infected intratracheally (no. 2) but in none of the foxes fed infected bird carcasses

Table

Mononuclear cells and alveolar epithelial cells in damaged parts of the lungs expressed the influenza virus antigen as diffuse red staining in their nucleus and, to a lesser extent, in their cytoplasm. Occasional cardiomyocytes in the periphery of a lesion in the heart expressed the influenza virus antigen as granular red staining in their nucleus. Lastly, neuronal and glial cells in the periphery of lesions in the cerebrum expressed the influenza virus antigen as granular to diffuse red staining in their nucleus and, to a lesser extent, their cytoplasm (Figure 2). No influenza virus antigen was detected in any cells of other organs, including the intestinal tract, of any other foxes.

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