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Peer-Reviewed Journal Tracking and Analyzing Disease Trends pages 1835–1988 EDITOR-IN-CHIEF D. Peter Drotman Managing Senior Editor EDITORIAL BOARD Polyxeni Potter, Atlanta, Georgia, USA Dennis Alexander, Addlestone Surrey, United Kingdom Senior Associate Editor Barry J. Beaty, Ft. Collins, Colorado, USA Martin J. Blaser, New York, New York, USA Brian W.J. Mahy, Atlanta, Georgia, USA Arturo Casadevall, New York, New York, USA Associate Editors Kenneth C. Castro, Atlanta, Georgia, USA Paul Arguin, Atlanta, Georgia, USA Thomas Cleary, Houston, Texas, USA Charles Ben Beard, Ft. Collins, Colorado, USA Anne DeGroot, Providence, Rhode Island, USA David Bell, Atlanta, Georgia, USA Vincent Deubel, Shanghai, China Charles H. Calisher, Ft. Collins, Colorado, USA Ed Eitzen, Washington, DC, USA Michael Drancourt, Marseille, France David Freedman, Birmingham, AL, USA Paul V. Effl er, Perth, Australia Kathleen Gensheimer, Augusta, ME, USA Peter Gerner-Smidt, Atlanta, GA USA Nina Marano, Atlanta, Georgia, USA Duane J. Gubler, Singapore Martin I. Meltzer, Atlanta, Georgia, USA Richard L. Guerrant, Charlottesville, Virginia, USA David Morens, Bethesda, Maryland, USA Scott Halstead, Arlington, Virginia, USA J. Glenn Morris, Gainesville, Florida, USA David L. Heymann, Geneva, Switzerland Patrice Nordmann, Paris, France Daniel B. Jernigan, Atlanta, Georgia, USA Tanja Popovic, Atlanta, Georgia, USA Charles King, Cleveland, Ohio, USA Jocelyn A. Rankin, Atlanta, Georgia, USA Keith Klugman, Atlanta, Georgia, USA Didier Raoult, Marseilles, France Takeshi Kurata, Tokyo, Japan Pierre Rollin, Atlanta, Georgia, USA S.K. Lam, Kuala Lumpur, Malaysia David Walker, Galveston, Texas, USA Bruce R. Levin, Atlanta, Georgia, USA David Warnock, Atlanta, Georgia, USA Myron Levine, Baltimore, Maryland, USA J. Todd Weber, Atlanta, Georgia, USA Stuart Levy, Boston, Massachusetts, USA Henrik C. Wegener, Copenhagen, Denmark John S. MacKenzie, Perth, Australia Marian McDonald, Atlanta, Georgia, USA Founding Editor John E. McGowan, Jr., Atlanta, Georgia, USA Joseph E. McDade, Rome, Georgia, USA K. Mills McNeill, Kampala, Uganda Copy Editors Tom Marrie, Edmonton, Alberta, Canada Ban Mishu-Allos, Nashville, Tennessee, USA Thomas Gryczan, Anne Mather, Beverly Merritt, Philip P. Mortimer, London, United Kingdom Carol Snarey, P. Lynne Stockton Fred A. Murphy, Galveston, Texas, USA Production Barbara E. Murray, Houston, Texas, USA Reginald Tucker, Ann Jordan, Shannon O’Connor, P. Keith Murray, Geelong, Australia Carrie Huntington Stephen M. Ostroff, Harrisburg, Pennsylvania, USA David H. Persing, Seattle, Washington, USA Editorial Assistant Richard Platt, Boston, Massachusetts, USA Susanne Justice Gabriel Rabinovich, Buenos Aires, Argentina Mario Raviglione, Geneva, Switzerland www.cdc.gov/eid Leslie Real, Atlanta, Georgia, USA David Relman, Palo Alto, California, USA Emerging Infectious Diseases Connie Schmaljohn, Frederick, Maryland, USA Emerging Infectious Diseases is published monthly by the Centers for Disease Control and Prevention, 1600 Clifton Road, Mailstop D61, Atlanta, GA 30333, Tom Schwan, Hamilton, Montana, USA USA. Telephone 404-639-1960, fax 404-639-1954, email [email protected]. Ira Schwartz, Valhalla, New York, USA Tom Shinnick, Atlanta, Georgia, USA The opinions expressed by authors contributing to this journal do not necessar- Bonnie Smoak, Bethesda, Maryland, USA ily refl ect the opinions of the Centers for Disease Control and Prevention or the Dixie E. Snider, Atlanta, Georgia, USA institutions with which the authors are affi liated. Rosemary Soave, New York, New York, USA All material published in Emerging Infectious Diseases is in the public domain Frank Sorvillo, Los Angeles, California, USA and may be used and reprinted without special permission; proper citation, how- P. Frederick Sparling, Chapel Hill, North Carolina, USA ever, is required. Robert Swanepoel, Johannesburg, South Africa Use of trade names is for identifi cation only and does not imply endorsement Phillip Tarr, St. Louis, Missouri, USA by the Public Health Service or by the U.S. Department of Health and Human Timothy Tucker, Cape Town, South Africa Services. Elaine Tuomanen, Memphis, Tennessee, USA John Ward, Atlanta, Georgia, USA ∞ Emerging Infectious Diseases is printed on acid-free paper that meets the requirements of ANSI/NISO 239.48-1992 (Permanence of Paper) Mary E. Wilson, Cambridge, Massachusetts, USA Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 12, December 2008 December 2008 On the Cover Surveillance of Coyotes to Detect Marc Chagall (1887–1985) Bovine Tuberculosis, Michigan .............1862 I and the Village (1911) K.C. VerCauteren et al. Oil on canvas (192.1 cm × 151.4 cm) Coyotes could be used as sentinels to detect Copyright ARS, NY. Mycobacterium bovis in the wild. Mrs. Simon Guggenheim Fund (146.1945) African Swine Fever Virus Digital Image Copyright the Museum of Modern Isolate, Georgia, 2007.............................1870 Art/Licensed by SCALA/Art Resource, NY. The Museum of Modern Art, New York, NY, USA R.J. Rowlands et al. The virus isolate, introduced to the Caucasus in 2007, is closely related to a group of viruses, About the Cover p. 1978 genotype II, circulating in Mozambique, Madagascar, and Zambia. CME ACTIVITY Clinical Characteristics and Molecular Subtyping of Research Vibrio vulnifi cus Illnesses, Israel ..........1875 R. Zaidenstein et al. Highly Pathogenic Avian Infl uenza The genetically distinct biotype 3 has penetrated Virus (H5N1) in Red Foxes Israeli freshwaters and is causing severe illness in Fed Infected Bird Carcasses .................1835 persons who handle tilapia or carp. L.A. Reperant et al. Foxes experimentally fed infected bird carcasses Dispatches excrete virus for up to 5 days without exhibiting severe disease and may thus disperse the virus. 1883 Novel Borna Virus in Psittacine Birds with Proventricular Dilatation Infl uenza Infection in Disease Wild Raccoons ........................................1842 p. 1838 K.S. Honkavuori et al. J.S. Hall et al. Raccoons can transmit avian and human infl uenza 1887 Possible Emergence of West and have a similar distribution of virus receptors in Caucasian Bat Virus in Africa respiratory tissues as humans. I.V. Kuzmin et al. 1890 Detection and Phylogenetic Analysis Enzootic Rabies Elimination of Group 1 Coronaviruses in South from Dogs and Reemergence in American Bats Wild Terrestrial Carnivores, p. 1889 C.V.F. Carrington et al. United States...........................................1849 A. Velasco-Villa et al. 1894 Rickettsia parkeri in Argentina Independent enzootics in wild terrestrial carnivores S. Nava et al. resulted from spillover events from long-term enzootics associated with dogs. 1898 Transmission of Atypical Bovine Prion Disease to Mice Transgenic for Genetic Characterization of Human Prion Protein Toggenburg Orbivirus, a New V. Béringue et al. Bluetongue Virus, Switzerland ..............1855 1902 Human Illnesses Caused by M.A. Hofmann et al. Opisthorchis felineus Flukes, Italy Nucleotide sequence analysis indicates that this O. Armignacco et al. virus is a new serotype of bluetongue virus. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 12, December 2008 1906 New Rabies Virus Variant in Mexican Immigrant A. Velasco-Villa et al. December 2008 1909 Avian Infl uenza Outbreaks in 1951 Human Case of Bartonella alsatica Chickens, Bangladesh Lymphadenitis P.K. Biswas et al. 1953 Ehrlichia chaffeensis in Amblyomma 1913 Outbreak of Trichinellosis Caused by parvum Ticks, Argentina Trichinella papuae, Thailand, 2006 1955 Enzootic Angiostrongyliasis in C. Khumjui et al. Shenzhen, China 1916 Multicenter Study of Brucellosis in 1956 Knowledge about Avian Infl uenza, Egypt European Region H. Samaha et al. 1957 Human Salmonella Infection Yielding 1919 Mycobacterium bovis Infection in CTX-M β-Lactamase, United States Holstein Friesian Cattle, Iran K. Tadayon et al. 1959 Yersinia pseudotuberculosis O:1 and Raw Carrots, Finland 1922 Hemoplasma Infection in HIV-positive Patient, Brazil 1961 Antibodies against Rickettsia spp. in p. 1894 A.P. dos Santos et al. Hunters, Germany 1925 Exposure to Streptococcus suis 1963 Rickettsia sp. in Ixodes granulatus among US Swine Workers Ticks, Japan T.C. Smith et al. 1965 Sin Nombre Virus Infection in Deer 1928 Multiple Francisella tularensis Mice, California Subspecies and Clades, Tularemia 1966 Parachlamydia acanthamoebae and Outbreak, Utah Abortion in Small Ruminants J.M. Petersen et al. 1968 Candidate New Species of Kobuvirus 1931 Mycobacterium bovis Strains and in Porcine Hosts Human Tuberculosis, Southwest Ireland 1970 Human Rickettsia felis Infection, O. Ojo et al. Taiwan 1935 Francisella novicida Bacteremia, 1972 Bartonella spp. and Rickettsia felis in p. 1903 Thailand Fleas, Democratic Republic of Congo A. Leelaporn et al. 1974 Antibodies to Nipah or Nipah-like Viruses in Bats, China Photo Quiz Book Review 1939 EID Photo Quiz M. Schultz 1977 Campylobacter, 3rd Edition Letters News & Notes 1943 Bartonella henselae Antibodies after About the Cover Cat Bite 1978 Eye to Eye in the Village 1944 Fatal Israeli Spotted Fever after Mediterranean Cruise Online Conference Summaries 1946 Streptococcus suis Meningitis Healthcare Infections Associated with Care Without Animal Contact, Italy and Treatment of Humans and Animals 1948 Equine Herpesvirus Type 9 in Giraffe www.cdc.gov/EID/content/14/12/e1.htm with Encephalitis Inaugural Meeting of the Cysticercosis 1950 Human Rabies Case with Long Working Group in Europe Incubation, Australia www.cdc.gov/EID/content/14/12/e2.htm Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 12, December 2008 Highly Pathogenic Avian Infl uenza 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, and Thijs Kuiken Eating infected wild birds may put wild carnivores at not stated) (9); however, these animals did not show clini- high risk for infection with highly pathogenic avian infl u- cal signs of disease. Also, recently, outbreaks of equine enza (HPAI) virus (H5N1). To determine whether red foxes infl uenza virus (H3N8) infections resulted in respiratory (Vulpes vulpes) are susceptible to infection with HPAI virus disease in domestic dogs (10,11). In contrast, within the (H5N1), we infected 3 foxes intratracheally. They excreted past 5 years, HPAI viruses (H5N1) have infected and killed virus pharyngeally for 3–7 days at peak titers of 103.5–105.2 carnivores belonging to 7 species: captive tigers (Panthera median tissue culture infective dose (TCID ) per mL and 50 tigris) and leopards (P. pardus) (12,13); domestic cats (14– had severe pneumonia, myocarditis, and encephalitis. To 17); captive Owston’s palm civets (Chrotogale owstoni) determine whether foxes can become infected by the pre- (18); a domestic dog (19); a free-living stone marten (Mar- sumed natural route, we fed infected bird carcasses to 3 other red foxes. These foxes excreted virus pharyngeally tes foina) (20); and a free-living American mink (21). In for 3–5 days at peak titers of 104.2–104.5 TCID /mL, but only these species, the infection resulted in both respiratory and 50 mild or no pneumonia developed. This study demonstrates extrarespiratory lesions, demonstrating systemic infection that red foxes fed bird carcasses infected with HPAI virus beyond the respiratory system. The most frequently report- (H5N1) can excrete virus while remaining free of severe dis- ed clinical signs for all species were respiratory distress, ease, thereby potentially playing a role in virus dispersal. neurologic signs, or both. The sources of most HPAI virus (H5N1) infections Infl uenza A viruses rarely infect species of the order Car- in carnivores were traced to infected birds eaten by the nivora. However, since 2003, highly pathogenic avian animals (12–15,19). Until 2005, carnivores infected with infl uenza (HPAI) viruses of subtype H5N1 have infected a HPAI virus (H5N1) were either wild carnivores kept in wide range of carnivore species. Within the past 30 years, captivity or domestic carnivores that ate infected domestic and before the emergence of HPAI viruses (H5N1), 5 doc- or peridomestic birds (12–14,19). Since 2005, and after the umented outbreaks of infl uenza virus infections occurred spread of HPAI virus (H5N1) of the Qinghai sublineage in 2 carnivore species—the harbor seal (Phoca vitulina) (clade 2.2) outside Southeast Asia in poultry and wild bird (1–4), and the American mink (Mustela vison) (5). In both populations, carnivores infected with HPAI virus (H5N1) species, the infection resulted in respiratory disease. In ad- included for the fi rst time free-living wild carnivores, which dition, infl uenza virus infection has been detected by virus presumably ate infected wild birds (20,21). culture or serologic examination in other carnivores, name- The occurrence of HPAI viruses (H5N1) in wild bird ly, domestic dogs (Canis lupus familiaris) (6,7), domestic populations is likely to result in the exposure and infection cats (Felis catus) (8,9), and bears kept in captivity (species of free-living wild carnivore species. In particular, abundant and widespread species of wild carnivores that have Author affi liations: Princeton University, Princeton, New Jersey, opportunistic feeding habits and that feed on wild birds may USA (L.A. Reperant, A.P. Dobson); and Erasmus Medical Centre, be at high risk for exposure. The red fox (Vulpes vulpes) is Rotterdam, the Netherlands (G. van Amerongen, M.W.G. van de one of the most abundant and widespread species of wild Bildt, G.F. Rimmelzwaan, A.D.M.E. Osterhaus, T. Kuiken) carnivores in Eurasia. Partly because of rabies eradication DOI: 10.3201/eid1412.080470 (22,23), fox populations in western Europe have increased Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 12, December 2008 1835 RESEARCH drastically since the mid-1980s (e.g., >140% in Germany; in negatively pressurized isolation units. Two negative- 24). The red fox is also an opportunistic carnivore species control foxes (nos. 4 and 8) were housed separately in an and has a diverse diet, which includes small mammals and indoor enclosure. birds (24,25). Therefore, it may likely hunt or scavenge To determine susceptibility to infection, we infected 3 wild birds infected with HPAI viruses (H5N1). However, foxes (nos. 1–3) intratracheally with a clade 2.2 HPAI virus the susceptibility of this species to infection with infl uenza (H5N1). Each anesthetized fox received 2.5 × 104 TCID 50 viruses is unknown. of virus in a volume of 2.5 mL through a catheter. One In this study, we asked 2 questions: 1) Are red foxes anesthetized fox (no. 4) was sham-infected intratracheally susceptible to infection with a wild bird isolate of HPAI with 2.5 mL of PBS and served as a negative control. To virus (H5N1) from clade 2.2? and 2) Can red foxes become determine whether red foxes can become infected by the infected by the presumed natural route of infection, i.e., presumed natural route of infection, we fed infected birds after feeding on infected bird carcasses? To answer these to 3 foxes (nos. 5–7). The infected birds were 1-week-old questions, we experimentally assessed the excretion pattern chicks that had been infected intratracheally with 2.5 × 104 (based on route, duration, and concentration of virus excre- TCID of the HPAI virus (H5N1) in a volume of 0.5 mL. 50 tion) and pathogenicity (based on clinical signs, death rates, At 24 hours postinoculation, the chicks were euthanized and distribution of lesions and virus) of a wild bird isolate by cervical dislocation and fed to foxes nos. 5–7 (2 whole of clade 2.2 HPAI virus (H5N1) in red foxes infected intra- chicks/fox). Homogenates of liver, lung, kidney, and brain tracheally and in red foxes fed infected bird carcasses. from infected chicks contained 106.3 to >109.3 TCID /g tis- 50 sue; pharyngeal and cloacal swabs reached titers of 104.5 to Materials and Methods 107.2 TCID /mL. On the basis of the relative weight of the 50 lungs, liver, kidneys, and brain of 1-week-old chicks weigh- Virus Preparation ing 50 to 55 g (27,28), foxes fed 2 chick carcasses received A virus stock was prepared of infl uenza virus A/ a minimal titer of 1010 TCID . Virus titers in internal or- 50 whooper swan/Germany/R65-1/2006 (H5N1), which was gans of dead wild or domestic birds naturally infected with isolated from a wild whooper swan (Cygnus cygnus) found HPAI virus (H5N1) have been reported sparingly; howev- dead on Rügen Island, Germany, in February 2006. (The er, an article from Japan reported high virus titers, e.g., as isolate was kindly provided by Dr Martin Beer, Friedrich- high as 107.5 TCID /mL, in the lung of a naturally infected 50 Loeffl er-Institute, Greifswald–Insel Riems, Germany.) It large-billed crow (Corvus macrorhynchos) (29). High titers was propagated twice in MDCK cells and titrated accord- were also detected in internal organs of 10-week-old chick- ing to standard methods (26). The stock reached an infec- ens and in highly susceptible species of wild swans, geese, tious virus titer of 106.9 median tissue culture infectious dose and ducks that were experimentally infected with a clade (TCID ) per mL. It was then diluted in phosphate-buffered 2.2 HPAI virus (H5N1), e.g., whooper swans, mute swans 50 saline (PBS) to obtain a concentration of 104 TCID /mL. (Cygnus olor), bar-headed geese (Anser indicus), common 50 pochards (Aythya ferina), and tufted ducks (Aythya fu- Experimental Design ligula) (30–32). For example, virus titers in internal organs Eight juvenile (6–10 months of age) red foxes were of common pochards and tufted ducks infected with a low obtained from a control program involving the fox popula- dose of clade 2.2 HPAI virus (H5N1) reached >106 TCID / 50 tion in the Netherlands. All were negative for antibodies mL (32). Our experimental design thus likely reproduces against infl uenza viruses according to a commercially avail- natural exposure after ingestion of dead or moribund birds able nucleoprotein-based ELISA test (European Veterinary infected with the virus. Our negative control was 1 fox (no. Laboratory, Woerden, the Netherlands) and for antibodies 8) that was fed 2 whole chicks that had been sham infected against canine distemper virus according to a virus neutral- with PBS. ization assay. The foxes had been treated against helmint- Before inoculation and at 1, 2, 3, 5, and 7 days postin- hic infections with 50 mg of fenbendazole when they were oculation (dpi), all foxes were anesthetized with ketamine- 2.5 months old and with 22.7 mg of praziquantel, 22.7 mg medetomidine, after which they were weighed, and nasal, of pyrantel base as pyrantel pamoate, and 113.4 mg of feb- pharyngeal, and rectal swabs were collected and placed in antel 2 months later. One month before the start of the ex- 3 mL of virus transport medium (Hank’s balanced salt so- periment, transponders (Star-Oddi, Reykjavik, Iceland) that lution containing 10% glycerol, 200 U/mL penicillin, 200 record body temperature every 15 minutes were placed in μg/mL streptomycin, 100 U/mL polymyxin B sulfate, and the peritoneal cavity of each fox, after the animal had been 250 μg/mL gentamicin). Each day, foxes were observed anesthetized with intramuscular injections of ketamine (5 for clinical signs; observers were ≈2 m from the isolation mg/kg) and medetomidine (0.05 mg/kg). During the ex- units. At 7 dpi, all foxes were anesthetized with ketamine- periment, 6 foxes (nos. 1–3 and 5–7) were singly housed medetomidine and euthanized by exsanguination. Experi- 1836 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 12, December 2008 HPAI (H5N1) in Foxes Fed Infected Bird Carcasses mental procedures were approved by an independent ani- and rectal swabs from another (Figure 1). Foxes infected mal care and use committee. intratracheally excreted the virus through the pharynx from 1 dpi on, up to 3–7 dpi; peak titers of pharyngeal excre- Postmortem and Immunohistochemical Examinations tion were 103.5–105.2 TCID /mL at 1–3 dpi. Foxes that had 50 Necropsy examinations and tissue sample collection been fed infected bird carcasses excreted the virus through were performed according to a standard protocol. After fi x- the pharynx from 1 dpi on, up to 3–5 dpi; peak titers of ation in 10% neutral-buffered formalin and embedding in pharyngeal excretion were 104.2–104.5 TCID /mL at 1 dpi. 50 paraffi n, tissue sections were stained with hematoxylin and Student t test showed no signifi cant difference in the pat- eosin for histologic evaluation, or they were processed ac- terns of pharyngeal excretion between the 2 groups of foxes cording to an immunohistologic method that used a mono- according to areas under the curve (t = –0.667, df = 4, p clonal antibody against the nucleoprotein of infl uenza A = 0.54). No virus was isolated from any swabs from any virus as a primary antibody for detection of infl uenza viral negative-control foxes. antigen (33). Lung tissue of an experimentally infected cy- The virus was isolated from the trachea (102.6 TCID /g 50 nomolgus macaque (Macaca fascicularis) experimentally tissue) and lung (103.3 TCID /g) of 1 of 3 foxes infected 50 infected with infl uenza virus A/Hong Kong/156/97 (H5N1) intratracheally (no. 2), and from the tonsil (102 TCID /g) 50 served as a positive control. Negative controls were created of another fox infected intratracheally (no. 3). No virus was by omitting the primary antibody or replacing the primary isolated from any of the organs of the foxes fed infected antibody with an irrelevant antibody, immunoglobulin G2 bird carcasses or of the negative-control foxes. (clone 20102; R&D, Abingdon, UK). The following tissues were examined by these 2 methods: conjunctiva, nasal con- Gross Examination cha, nasal septum, trachea, lung (6 specimens/fox), tongue, Of the 3 foxes infected intratracheally, 2 (nos. 1 and 2) esophagus, stomach, duodenum, jejunum, ileum, cecum, had extensive multifocal or coalescing pulmonary lesions, colon, tonsil, tracheobronchial lymph node, mesenteric which were dark purple and slightly fi rm (Figure 2). The 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, Swit- zerland). Serial dilutions (10-fold) of the tissue homogenates and swabs were inoculated into MDCK cells in trip- licate as described previously (26). The minimal detectable titer was 100.8 TCID /mL. All experiments were performed 50 under BioSafety Level 3 conditions. Results Clinical Signs Clinical signs were not observed in foxes infected intratracheally or in foxes fed infected bird carcasses. How- ever, 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- Figure 1. Infectious virus titers obtained from pharyngeal, nasal, and control foxes (nos. 4 and 8). rectal swabs of foxes infected intratracheally with highly pathogenic avian infl uenza (HPAI) virus (H5N1) (left, black symbols) or fed chicks infected with HPAI virus (H5N1) (right, gray symbols) at Virology various time points after infection. No virus was isolated from any The virus was isolated from pharyngeal swabs from all swabs of the negative-control foxes. TCID , median tissue culture infected foxes and from nasal and rectal swabs from 1 fox 50 infectious dose. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 12, December 2008 1837 RESEARCH Figure 2. Lesions and associated expression of infl uenza 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. estimated percentage of affected lung tissue was 20% (no. stitial or interstitial pneumonia. They had small- to medi- 1) and 80% (no. 2). In contrast, 1 of the 3 foxes infected in- um-sized foci of infl ammation in the lungs, located mostly tratracheally (no. 3) and all foxes fed infected bird carcasses around the bronchioles and characterized by thickened had >2 small multifocal lesions (1–5 mm), which affected alveolar walls that were infi ltrated with macrophages and <5% of the lungs. In addition, 2 of the 3 foxes fed infected neutrophils. Type II pneumocyte and bronchiolar epithelial bird carcasses (nos. 5 and 6) had randomly distributed pete- hyperplasia was observed in the lungs of fox no. 7. Respira- chial hemorrhages throughout the lungs. Moderate enlarge- tory organs of negative-control foxes had no lesions. ment of the spleen, tonsils, and/or tracheobronchial lymph Extrarespiratory histologic lesions were seen only in nodes was observed in all foxes, whether infected intra- foxes infected intratracheally, namely, in the heart of fox tracheally or fed infected bird carcasses. Negative-control no. 2 and in the cerebrum of foxes nos. 1 and 2. Fox no. 2 foxes had no respiratory or extrarespiratory lesions. had multiple infl ammatory and necrotic lesions in the myo- cardium, characterized by infi ltration of macrophages and Histopathologic Findings neutrophils and necrotic cardiomyocytes (Figure 2). The ce- Histologic lesions were found in foxes infected intratra- rebrum of foxes nos. 1 and 2, infected intratracheally, had cheally and in foxes fed infected bird carcasses. However, multiple lesions of acute to subacute encephalitis, from mild the lesions were more severe in foxes infected intratrache- to severe, characterized by perivascular cuffi ng, foci of glio- ally (Table). The 2 most severely affected foxes (nos. 1 and sis or neuronal necrosis, or a combination of these lesions; 2, infected intratracheally) had severe hemorrhagic bron- their cerebellum and brain stem did not show any lesions chointerstitial pneumonia with extensive coalescing lesions (Figure 2). No relevant lesions were seen in other organs, of infl ammation and necrosis, characterized by macrophage including organs of the digestive tract, of any other foxes. and neutrophil infi ltration of the alveolar walls and loss of histologic architecture. The alveolar and bronchiolar lumina Immunohistochemical Findings were fi lled with alveolar macrophages, neutrophils, and Cells expressing the infl uenza virus antigen were pres- erythrocytes, mixed with fi brin and cellular debris. In both ent in the lungs, heart, and brain of 1 of 3 foxes infected foxes, sloughing of the alveolar and bronchiolar epithelia intratracheally (no. 2) but in none of the foxes fed infected indicated necrosis, and type II pneumocyte and bronchiolar bird carcasses (Table). Mononuclear cells and alveolar epithelial hyperplasia indicated regeneration (Figure 2). The epithelial cells in damaged parts of the lungs expressed other foxes (no. 3, infected intratracheally, and all foxes fed the infl uenza virus antigen as diffuse red staining in their infected bird carcasses) had minimal to mild bronchointer- nucleus and, to a lesser extent, in their cytoplasm. Occa- 1838 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 12, December 2008 HPAI (H5N1) in Foxes Fed Infected Bird Carcasses Table. Distribution of lesions and influenza virus antigen expression in experimentally infected red foxes* Lesions Influenza virus antigen Route of inoculation, fox no. Lungs Heart Brain Lungs Heart Brain Intratracheal inoculation 1 +++ – + – – – 2 +++ + ++ ++ + +++ 3 ++ – – – – – Fed infected bird carcasses 5 + – – – – – 6 – – – – – – 7 + – – – – – *Foxes were infected either intratracheally with highly pathogenic avian influenza (HPAI) virus (H5N1) or by being fed chicks infected with HPAI virus (H5N1); they were examined at 7 days postinoculation. Respiratory lesions, extrarespiratory lesions, or influenza virus antigen expression were not observed in negative-control foxes. –, absence of lesions (no cells expressing the influenza virus antigen); +, mild and focal or multifocal lesions (few cells expressing the influenza virus antigen); ++, moderate and multifocal lesions (moderate number of cells expressing the influenza virus antigen); +++, severe and extensive lesions (numerous cells expressing the influenza virus antigen). sional cardiomyocytes in the periphery of a lesion in the cephalitis may develop in those inoculated intratracheally. heart expressed the infl uenza virus antigen as granular red Frequent fi ndings of HPAI virus (H5N1) infections in natu- staining in their nucleus. Lastly, neuronal and glial cells rally infected carnivores were pneumonia associated with in the periphery of lesions in the cerebrum expressed the respiratory distress and encephalitis (in some cases associ- infl uenza virus antigen as granular to diffuse red staining in ated with neurologic signs) (12–14,16,18–21). In most in- their nucleus and, to a lesser extent, their cytoplasm (Fig- stances, the animals were either euthanized because of the ure 2). No infl uenza virus antigen was detected in any cells severity of the disease or were found dead. Surprisingly, of other organs, including the intestinal tract, of any other foxes with severe respiratory and cerebral lesions did not foxes. show any visible clinical signs. Foxes, being wild animals, were wary in the presence of humans and changed their be- Discussion havior even when observed from a distance. This behavior This study demonstrates that red foxes are susceptible may have prevented us from observing subtle clinical signs, to infection with a wild bird isolate of HPAI virus (H5N1) notably abnormal breathing. A cat that died of HPAI virus from clade 2.2. Red foxes can become infected after eating (H5N1) infection in Germany did not show visible clini- infected bird carcasses, and they can excrete the virus for cal signs 24 hours before death, despite marked respiratory as many as 5 days in the absence of severe disease. There- lesions (16), which suggests that even severe respiratory fore, naturally infected red foxes may potentially survive lesions may not be noticed clinically. Clinical manifesta- infection in the wild and excrete and disperse HPAI viruses tions of neurologic lesions in infected foxes may have gone (H5N1) within their home ranges. The size of foxes’ home unnoticed because the lesions were in the cerebrum rather ranges depends on the environmental conditions and avail- than in the cerebellum. Although cerebellar lesions may ability of food resources, but typically it varies between cause conspicuous neurologic signs, e.g., ataxia and loss 1 km² and 10 km² (34). Red foxes are highly mobile and of balance, cerebral lesions may cause more subtle clinical may travel 5–20 km within their home range during a night signs, e.g., altered mental attitude, which were not noticed (35). A juvenile fox traveled 90 km in 1 direction within 5 under these experimental conditions (36). days during fall dispersal from its place of birth (35). Fur- Foxes may exhibit more severe disease after eating thermore, red foxes have colonized most urbanized areas infected birds under natural conditions than under the con- in Europe, resulting in increased contact with domestic and trolled conditions of our feeding experiments, because of peridomestic animals (23). They may transmit the virus to poorer health, possible co-infections, and poorer nutritional domestic species, such as poultry. Therefore, we propose status of wild animals. For instance, the cats that died of that this abundant and widespread carnivorous species be HPAI virus (H5N1) infection in Germany were all infected surveyed for exposure to or infection with HPAI viruses with Aelurostrongylus spp., and pulmonary aelurostrongy- (H5N1) in infl uenza-endemic areas or in areas experienc- losis was considered to have contributed to the severity of ing outbreaks of HPAI virus (H5N1) infections in wild bird the disease in these animals (16). Fatal cases of HPAI virus populations. Where foxes are hunted, carcasses may be (H5N1) infection in red foxes may have been missed after routinely sampled and tested. Where foxes are protected or the spread of HPAI viruses (H5N1) in poultry and wild bird not hunted, live trapping, bleeding, and pharyngeal swab- populations outside Asia because fox carcasses are diffi cult bing of anesthetized foxes may be implemented. to locate and because those found may likely be routinely Although red foxes fed infected bird carcasses may tested for rabies and canine distemper rather than for infl u- survive infection, severe pneumonia, myocarditis, and en- enza virus infection. Therefore, we suggest that red foxes Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 12, December 2008 1839 RESEARCH with neurologic signs or red foxes found moribund or dead References in disease-endemic areas or in areas experiencing outbreaks 1. Lang G, Gagnon A, Geraci JR. 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