Systemic dissemination of H5N1 influenza A viruses in ferrets and hamsters after direct intragastric inoculation

Abstract

Although oral exposure to H5N1 highly pathogenic avian influenza viruses is a risk factor for infection in humans, it is unclear how oral exposure to these virus results in lethal respiratory infections. To address this issue, we inoculated ferrets and hamsters with two highly pathogenic H5N1 strains. These viruses, inoculated directly into the stomach, were isolated from the large intestine and the mesenteric lymph nodes within 1 day of inoculation and subsequently spread to multiple tissues, including lung, liver, and brain. Histopathologic analysis of ferrets infected with virus via direct intragastric inoculation revealed lymph folliculitis in the digestive tract and mesenteric lymph nodes and focal interstitial pneumonia. Comparable results were obtained with the hamster model. We conclude that, in mammals, ingested H5N1 influenza viruses can disseminate to nondigestive organs, possibly through the lymphatic system of the gastrointestinal tract.

Fig. 1.

Gastric delivery of gelatin capsules in ferrets. To ensure that the gelatin capsules remained intact until they reach the stomach, we coated their interiors with petrolatum (Vaseline), filled them with barium (0.4 g/capsule), and allowed the ferrets to ingest them (four capsules/animal). Lateral views 5 (a) and 30 (b) min after oral ingestion of the capsules are shown. Note that the barium (white) did not leak into the esophagus on release, but rather flowed through the digestive tract.

Fig. 2.

Histopathologic findings in ferrets infected with the UT3062 virus via the digestive route. (a) Folliculitis with viral antigens (arrows) in the intestinal lymph follicles (F; Peyer's patches) in a ferret on day 1 postinfection (p.i.) (bar = 500 μm). Asterisk, lumen of intestine. (b) Mesenteric lymphadenitis with viral antigens (brown pigment) on day 1 p.i. (bar = 50 μm). (c) Viral antigens detected on day 3 p.i. in the tonsilar lesions (brown pigments) of a ferret infected via the intragastric route (bar = 1 mm).

Fig. 3.

Gastric inoculation of hamsters. We performed temporary percutaneous gastrostomy, as described in the text, and injected barium (0.5 ml/animal) directly into the stomach percutaneously using a 23-guage needle and 1-ml syringe. The injected barium (white) did not leak into the abdominal space, but rather flowed through the digestive tract. (a) Five minutes after gastric inoculation of barium (lateral view). (b) Thirty minutes after gastric administration of barium (lateral view).

Fig. 4.

Histopathologic findings in hamsters infected with the UT3062 virus. (a) Lung specimen from an intranasally infected hamster showing bronchopneumonia (bar = 500 μm) with viral antigens (brown pigment) in bronchiolar epithelia at day 1 p.i. (inset bar = 50 μm). Asterisks, lumen of bronchus; v, blood vessels. (b) Lung specimen from hamster infected via the intragastric route showing severe interstitial pneumonia with extensive thickening of the alveolar wall on day 3 p.i. (bar = 50 μm). (Inset) Bronchiolar epithelium showed neither obvious pathological changes nor viral antigens on day 3 p.i. (bar = 50 μm). (c) Lung tissue from a control hamster showing thin alveolar walls and no inflammatory infiltrates (bar = 50 μm).

Fig. 5.

Illustration of how ingested viruses could reach the respiratory organs from the intestine. Pink represents arterial blood flow, blue the venous route, and green the lymphatic route. (a) Ingested food containing infectious virus (red) reaches the intestine. (b) The virus invades the lymph nodes, causing lymphadenitis. (c) Lymph fluid with virus particles flows into the thoracic duct, which connects to the venous route (red arrow). (e) The virus particles enter the venous route and, ultimately, the right side of the heart and the pulmonary circulation. When they reach the lungs, they cause pneumonia.