Field Sample Collection

Blood was opportunistically collected from wild raccoons taken during population control operations in various counties/parishes in Texas, Wyoming, Louisiana, California, and Maryland. Raccoons from northwestern Georgia were sampled as part of the US Department of Agriculture Cooperative National Oral Rabies Vaccination program. Raccoons in Colorado were captured for this, and other studies, in and around Fort Collins, Colorado. Blood samples were obtained by cardiac or jugular puncture, allowed to clot, and centrifuged to separate serum from cellular blood components. Serum was transferred to fresh cryovials and stored frozen (–20°C) until transport to the National Wildlife Research Center in Fort Collins, where they were stored at –80°C until analysis.

Screening for Antibodies to Influenza Virus

Agar gel immunodiffusion is a serologic assay used to detect antibodies to influenza viruses. The antigen used in the assay was derived from the matrix and nucleoproteins of AI and is used to detect antibodies to all subtypes of AI. The procedure has been described by Beard (6) and was performed by using reagents and the protocol provided by the Center for Veterinary Biologics and National Veterinary Services Laboratories (Ames, IA, USA).

Determination of Antibody Subtypes for Influenza Virus

Hemagglutination inhibition and neuraminidase inhibition are used to determine subtype identity of influenza antibodies in sera. These procedures are described by Beard (6) and were performed at the National Veterinary Services Laboratories.

Experimental Infection of Raccoons

Ten wild raccoons were live-trapped in and around Fort Collins, Colorado. These animals were transported to the National Wildlife Research Center and held for a 2-week quarantine period, where they were observed daily and judged to be in good overall health on the basis of food intake, behavior, and absence of clinical signs of disease. After 2 weeks, the animals were anesthetized and moved into a biocontainment level 2 facility. Blood samples and nasal and rectal swabs were collected, and animals were placed in individual cages (5 per room). Four animals in each room were inoculated intranasally with 10⁵ 50% egg infectious dose (EID₅₀) of AI virus A/CK/AL/75 (H4N8) diluted in 100 μL of viral transport medium. The fifth animal in each room was uninoculated and monitored to determine transmission between animals. To prevent potential fomite transmission, these controls were always handled and sampled first when technicians entered the rooms, and all food and water bowls were cleaned and sanitized in hypochlorite solution each day. Each animal was provided food and water ad libitum and observed daily for illness, behavior, and general welfare. To avoid excessive handling and anesthesia, serum samples, nasal and rectal swabs, and rectal temperatures were collected from each group of 5 raccoons on alternating days for 14 days. Blood samples were obtained by jugular puncture. Nasal and rectal swabs were collected by using dacron-tipped applicators placed into viral transport media after swabbing. All samples were stored at –80°C until analyzed.

Subsequently, a second cohort of 6 raccoons was captured in Fort Collins, quarantined, and placed into biocontainment. Four raccoons were intranasally inoculated with 10⁵ EID₅₀ of human influenza virus (A/Aichi/2/68 [H3N2]) and were sampled and monitored as described above. Two uninoculated raccoons were housed in cages adjacent to inoculated animals to assess transmission. All animal handling, trapping, and experimental infections were performed following Institutional Animal Care and Use Committee and institutional biosafety protocols, guidelines, and approval.

Reverse Transcription–PCR

Viral RNA was extracted from nasal swabs by using the QIAamp Viral RNA Mini Kit (QIAGEN, Valencia, CA, USA), following manufacturer’s instructions. Viral RNA from rectal swabs was extracted by using the same procedure with addition of half of an Inhibitex tablet (Stool Extraction Kit; QIAGEN) to remove PCR inhibitors. Real-time reverse transcription–PCR (RT-PCR) was performed following the procedure of Spackman et al. (7). Samples were compared with standard curves generated from known concentrations of AI, extracted, and amplified by using the same procedures. Results are expressed as EID₅₀ equivalents. These procedures were used to analyze environmental samples (feces and water) as part of the national surveillance for highly pathogenic AI (8).

Detection of Influenza Virus Cellular Receptors in Raccoon Respiratory Tissues

Airway tissue sections were collected from 5 adult raccoons (humanely killed in a different study) from 7 standardized locations (nasal mucosa; larynx; upper, middle, and lower trachea; bronchus; lung), fixed in formalin, and embedded in paraffin. Airway tissues were cut into 5 μm–thick sections, mounted on 3-aminopropyltrethoxy-silane–coated slides, deparaffinized in xylene, and rehydrated in alcohol. For detection of sialic acids (SAs), sections were stained with SAα2,3Gal- and SAα2,6Gal-specific lectins. Briefly, sections were incubated overnight with 250 μL of Western blocking solution (Roche Biochemicals, Indianapolis, IN, USA), washed 3× in Tris-buffered saline, pH 7.6, and incubated with 250 μL of fluorescein isothiocyanate–labeled Sambucus nigra lectin (Vector Laboratories, Burlingame, CA, USA) and biotinylated Maackia amurensis lectin (Vector Laboratories) overnight at 4°C. After 3 washes in Tris-buffered saline, sections were incubated with Alexa Fluor 594–conjugated streptavidin (Molecular Probes, Inc., Eugene, OR, USA) for 2 hours at room temperature. The sections were washed, counterstained with 4′,6-diamino-2-phenylindole dihydrochloride (Molecular Probes), washed again, and mounted on cover glass. Sections were examined with a fluorescence microscope (Carl Zeiss, Inc., Oberkochen, Germany). Because previous research has demonstrated that equine tracheal epithelial cells predominantly express SAα2,3Gal residues and pig tracheal cells express SAα2,3Gal and SAα2,6Gal residues (2), sections of equine and porcine trachea were included as positive controls for each staining procedure.

Serologic Survey of Wild Raccoons for Exposure to AI Virus

We screened 730 wild raccoons from California, Texas, Louisiana, Maryland, Wyoming, and Colorado. Of these, 17 (2.4%) had antibodies to AI virus. Table 1 summarizes the raccoon serosurvey and subtyping results from these states. Four (2.4%) of 168 Maryland raccoons in 2004 had antibodies to AI virus with 3 hemagglutinin subtypes represented. Two of these raccoons had antibodies to 2 subtypes, which indicated multiple exposures to AI virus. Colorado and Wyoming also had seropositive raccoons with prevalences of 12.8% and 25%, respectively. Multiple subtypes were present in both populations, and multiple exposures in individual raccoons were observed. However, none of the raccoons from Georgia, Texas, or California showed serologic evidence of exposure to AI virus. These results indicated that wild raccoons are exposed to a variety of AI virus subtypes and seroconvert on the basis of these exposures.

Experimental Infection of Raccoons with AI Virus

To determine whether raccoons are competent hosts for AI virus infection and are capable of shedding and transmitting virus, raccoons were infected with a specific subtype of AI virus (H4N8) and monitored for symptoms of infection and disease. Two of 10 wild-caught raccoons had antibodies to AI virus (Table 1). These animals were included in the infection study because the AI virus inoculum used was a different subtype, but with potential cross-neutralization as a caveat.

Eight raccoons were inoculated intranasally with 10^5.0 EID₅₀ of AI virus (H4N8) and monitored for 14 days postinoculation (dpi). Four (50%) of these animals became infected, as shown by nasal shedding of viral RNA detected by RT-PCR. Two of these animals (256 and 275) shed detectable amounts of virus at only 1 time point (1 dpi). Another raccoon (259) shed virus at least up to 6 dpi, and the other infected raccoon (263) shed for the entire 14 days of the study (Table 2). RT-PCR analyses of rectal swabs showed no detectable viral RNA shed by digestive tracts of infected raccoons (data not shown), which is consistent with influenza being primarily a respiratory disease in mammals (2).

One of the 2 uninoculated raccoons housed in cages adjacent (within ≈0.5 m) to inoculated raccoons developed nasal shedding of virus. Every precaution was taken to prevent inadvertent transmission by handling; thus, this animal (262) probably contracted the virus by aerosol from >1 of its infected cohorts. This result indicated that raccoons are capable of transmitting influenza virus from one to another. Given the small amounts of AI virus shed by these raccoons and the timing of infection of this animal, we cannot rule out possible aerosolization of inoculum by adjacent raccoons and transmission by that route.

Three of the 5 raccoons that shed virus developed antibodies to the AI virus (H4N8) isolate, including raccoon 262, which was not inoculated but contracted the virus from adjacent, infected raccoons (Table 3). Raccoon 259 was humanely killed on 8 dpi because of an unrelated physical condition (tooth abscess), presumably before detectable antibodies were produced. Raccoon 256 shed virus only on 1 dpi yet developed detectable antibodies to AI virus (H4N8) by 9 dpi. However, the other raccoon that shed virus on 1 dpi (275) did not develop a detectable immune response, which indicated that virus detected in the swab was probably residual inoculum. Raccoons 263 and 262 had preexisting antibodies to a different subtype of AI virus that did not prevent infection and seroconversion to the other AI virus (H4N8) inoculum.

We observed no overt clinical signs of disease in these animals. Rectal temperatures showed no obvious trends and were probably confounded by stresses of anesthesia and handling. Most of the animals appeared lethargic, possibly because of confinement and manipulations occurring during daytime (raccoons are nocturnal). All other animals ate and drank well and most gained weight over the course of the experiment (data not shown).

Influenza Virus Receptors in Raccoons

Figure

Figure. Raccoon respiratory tissues stained with lectins specific for sialic acids (SAs) with α2,6- and α2,3-linkages. A) Upper trachea; B) lower trachea; C) bronchus; D) bronchiole. Arrows indicate endothelial lining of the...

The predominant receptor for AI virus is SA linked α2,3 to galactose. In waterfowl, these receptors are located primarily in intestinal epithelium, which is why AI is primarily a disease of the digestive tract in avian species. In contrast, humans have SA linked α2,6 to galactose that is located predominantly in the respiratory system (2). Tissues from raccoon respiratory tracts were examined for avian and human influenza virus receptors by staining with lectins specific for each type of receptor (Figure). Raccoons have both receptor types in their respiratory systems, similar to swine but with uneven distribution among tissues. In the upper trachea epithelium, the overwhelmingly predominant receptor is the human type SAα2,6 (Figure, panel A). As one examines tissues from deeper in the respiratory tract, increasing amounts of the avian SA α2,3 receptor are found until the 2 types of receptors are in roughly equal amounts in the lungs (Figure, panels B, C, and D).

Experimental Infection of Raccoons with Human Influenza Virus

The presence and distribution of human type receptors in raccoons led us to infect a new cohort of raccoons with human influenza virus (H3N2). Daily monitoring showed that inoculated animals shed virus nasally for up to 8 dpi (Table 4, Figure). The amounts of virus shed were larger than in the AI experimental infection study but no transmission to either co-housed, virus-free raccoon was detected. All 4 inoculated animals subsequently developed antibodies against this virus by 14 dpi (data not shown). One raccoon (272) shed small amounts of virus rectally (0.25 EID₅₀ equivalents) on 5 dpi, but no other rectal shedding of virus was detected. As with AI virus infection, no obvious clinical signs of disease were observed in these animals. Infected raccoons were also capable of shedding moderate amounts of human influenza virus, although no transmission to virus-free animals was observed.