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


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

In This Article

Materials and Methods

Virus Preparation

A virus stock was prepared of influenza virus A/whooper swan/Germany/R65-1/2006 (H5N1), which was isolated from a wild whooper swan (Cygnus cygnus) found dead on Rügen Island, Germany, in February 2006. (The isolate was kindly provided by Dr Martin Beer, Friedrich-Loeffler-Institute, Greifswald-Insel Riems, Germany.) It was propagated twice in MDCK cells and titrated according to standard methods.[26] The stock reached an infectious virus titer of 106.9 median tissue culture infectious dose (TCID50) per mL. It was then diluted in phosphate-buffered saline (PBS) to obtain a concentration of 104 TCID50/mL.

Experimental Design

Eight juvenile (6-10 months of age) red foxes were obtained from a control program involving the fox population in the Netherlands. All were negative for antibodies against influenza viruses according to a commercially available nucleoprotein-based ELISA test (European Veterinary Laboratory, Woerden, the Netherlands) and for antibodies against canine distemper virus according to a virus neutralization assay. The foxes had been treated against helminthic infections with 50 mg of fenbendazole when they were 2.5 months old and with 22.7 mg of praziquantel, 22.7 mg of pyrantel base as pyrantel pamoate, and 113.4 mg of febantel 2 months later. One month before the start of the experiment, transponders (Star-Oddi, Reykjavik, Iceland) that record body temperature every 15 minutes were placed in the peritoneal cavity of each fox, after the animal had been anesthetized with intramuscular injections of ketamine (5 mg/kg) and medetomidine (0.05 mg/kg). During the experiment, 6 foxes (nos. 1-3 and 5-7) were singly housed in negatively pressurized isolation units. Two negative-control foxes (nos. 4 and 8) were housed separately in an indoor enclosure.

To determine susceptibility to infection, we infected 3 foxes (nos. 1-3) intratracheally with a clade 2.2 HPAI virus (H5N1). Each anesthetized fox received 2.5 × 104 TCID50 of virus in a volume of 2.5 mL through a catheter. One anesthetized fox (no. 4) was sham-infected intratracheally with 2.5 mL of PBS and served as a negative control. To determine whether red foxes can become infected by the presumed natural route of infection, we fed infected birds to 3 foxes (nos. 5-7). The infected birds were 1-week-old chicks that had been infected intratracheally with 2.5 × 104 TCID50 of the HPAI virus (H5N1) in a volume of 0.5 mL. At 24 hours postinoculation, the chicks were euthanized by cervical dislocation and fed to foxes nos. 5-7 (2 whole chicks/fox). Homogenates of liver, lung, kidney, and brain from infected chicks contained 106.3 to >109.3 TCID50/g tissue; pharyngeal and cloacal swabs reached titers of 104.5 to 107.2 TCID50/mL. On the basis of the relative weight of the lungs, liver, kidneys, and brain of 1-week-old chicks weighing 50 to 55 g,[27,28] foxes fed 2 chick carcasses received a minimal titer of 1010 TCID50. Virus titers in internal organs of dead wild or domestic birds naturally infected with HPAI virus (H5N1) have been reported sparingly; however, an article from Japan reported high virus titers, e.g., as high as 107.5 TCID50/mL, in the lung of a naturally infected large-billed crow (Corvus macrorhynchos).[29] High titers were also detected in internal organs of 10-week-old chickens and in highly susceptible species of wild swans, geese, and ducks that were experimentally infected with a clade 2.2 HPAI virus (H5N1), e.g., whooper swans, mute swans (Cygnus olor), bar-headed geese (Anser indicus), common pochards (Aythya ferina), and tufted ducks (Aythya fuligula).[30,31,32] For example, virus titers in internal organs of common pochards and tufted ducks infected with a low dose of clade 2.2 HPAI virus (H5N1) reached >106 TCID50/mL.[32] Our experimental design thus likely reproduces natural exposure after ingestion of dead or moribund birds infected with the virus. Our negative control was 1 fox (no. 8) that was fed 2 whole chicks that had been sham infected with PBS.

Before inoculation and at 1, 2, 3, 5, and 7 days postinoculation (dpi), all foxes were anesthetized with ketaminemedetomidine, after which they were weighed, and nasal, pharyngeal, and rectal swabs were collected and placed in 3 mL of virus transport medium (Hank's balanced salt solution containing 10% glycerol, 200 U/mL penicillin, 200 μg/mL streptomycin, 100 U/mL polymyxin B sulfate, and 250 μg/mL gentamicin). Each day, foxes were observed for clinical signs; observers were ≈2 m from the isolation units. At 7 dpi, all foxes were anesthetized with ketamine-medetomidine and euthanized by exsanguination. Experimental procedures were approved by an independent animal care and use committee.

Postmortem and Immunohistochemical Examinations

Necropsy examinations and tissue sample collection were performed according to a standard protocol. After fixation in 10% neutral-buffered formalin and embedding in paraffin, tissue sections were stained with hematoxylin and eosin for histologic evaluation, or they were processed according to an immunohistologic method that used a monoclonal antibody against the nucleoprotein of influenza A virus as a primary antibody for detection of influenza viral antigen.[33] Lung tissue of an experimentally infected cynomolgus macaque (Macaca fascicularis) experimentally infected with influenza virus A/Hong Kong/156/97 (H5N1) served as a positive control. Negative controls were created by omitting the primary antibody or replacing the primary antibody with an irrelevant antibody, immunoglobulin G2 (clone 20102; R&D, Abingdon, UK). The following tissues were examined by these 2 methods: conjunctiva, nasal concha, nasal septum, trachea, lung (6 specimens/fox), tongue, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, tonsil, tracheobronchial lymph node, mesenteric lymph node, spleen, thymus, heart, liver, pancreas, kidney, adrenal gland, urinary bladder, olfactory bulb, cerebrum (at level of hippocampus), cerebellum, and brain stem.

Virus Titrations

The same tissues examined for histopathologic changes were also sampled for viral titration. Tissue samples were weighed and homogenized in 3 mL of transport medium with a homogenizer (Kinematica Polytron, Lucerne, Switzerland). Serial dilutions (10-fold) of the tissue homogenates and swabs were inoculated into MDCK cells in triplicate as described previously.[26] The minimal detectable titer was 100.8 TCID50/mL. All experiments were performed under BioSafety Level 3 conditions.


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