Subclinical Infection With Avian Influenza A (H5N1) Virus in Cats

Michael Leschnik; Joachim Weikel; Karin Möstl; Sandra Revilla-Fernández; Eveline Wodak; Zoltan Bagó; Elisabeth Vanek; Viviane Benetka; Michael Hess; Johann G. Thalhammer


Emerging Infectious Diseases. 2007;13(2):243-247. 

In This Article

Materials and Methods

Pharyngeal swabs of 40 cats were sampled[10] on day 8 and tested for H5N1 virus by PCR; positive results were obtained for 3 cats (cats 1, 2, and 3). All positive results were confirmed at the OIE reference laboratory in Weybridge, United Kingdom. All PCRs for H5N1 were conducted at the Agency for Health and Food Safety in Mödling, Austria. Daily physical examination by veterinarians showed no signs of influenza in any cat on days 4-21. In a follow-up examination on day 15, 0 of 34 cats of the 40 cats previously tested (on day 8) were positive for H5N1 virus in pharyngeal swabs. In 3 cats that had died during this period, necropsy showed no evidence of infectious respiratory disease, and PCR results for influenza virus were negative.

On day 22 after the H5N1-infected swan was put in the animal shelter, 167 cats (5 kittens 4-6 months of age and 162 adults) were still available for further observations. Three cats had died and 24 other cats had been placed in private households. Before discharge from the shelter and within 1 week thereafter, all of these cats were examined and no abnormal health status was observed.

A total of 167 cats were transported in small groups in ≈50 containers for 12 h from the animal shelter to a quarantine area and housed in 2 separate groups from day 22 until day 50. Average floor space for each cat was ≈1.4m2. The larger group contained 139 cats (including cats 1 and 2); the smaller group contained 28 cats. Cat 3 was not available for further examination because it was healthy before leaving the shelter and, to our knowledge, did not die. The smaller group was always separated from the larger group and was kept indoors at the animal shelter in Graz. In the quarantine area, the 167 cats were housed in 2 closed rooms, without any activity restriction, and had free access to food and water. Routine physical examination, including auscultation of the chest, was done on days 22, 29, and 50 for all cats at the quarantine station. In case of an obvious health problem, clinical signs were monitored by daily physical examination and serologic testing. The litter pans of the cats and floors of the quarantine areas were cleaned every day and disinfected every other day.

On days 22 and 29, pharyngeal and rectal swabs were obtained and transported in phosphate-buffered saline containing antimicrobial drugs.[10] Swabs were obtained with special care to avoid any contact with the environment and were transferred immediately into tubes containing transport media. Blood was obtained on days 22, 29, 36, and 50. To facilitate physical examinations and collection of samples, we gave mild general anesthesia (propofol and midazolam) to all cats on day 29 ( Table 1 ).

Pharyngeal and rectal swabs were examined for the matrix gene of influenza A virus by using a real-time reverse transcription-PCR (RT-PCR) according to the method of Spackman et al..[11] To screen for additional infections that might influence the health and immune status of the cats, we obtained 64 additional pharyngeal swabs on day 29 from cats with upper respiratory symptoms and tested them for nucleic acids specific for feline herpesvirus 1 (FHV-1)- and feline calicivirus (FCV). Real-time RT-PCR for FCV was conducted in a volume of 25 µL (22 µL reaction mixture and 3 µL template) in the Real-Time PCR system 7300 (Applied Biosystems, Foster City, CA, USA). The reaction mixture was prepared following the manufacturer's instructions of a commercially available kit (SuperScript III Platinum One-Step Quantitative RT-PCR Kit, Invitrogen, Carlsbad, CA , USA). This mixture contained 10 pmol/L of each primer (forward primer: 5´-AGTGGCATGACCGCCCT-3´, reverse primer: 5´-CGTTAGCGCAGGTTGAGCA-3´), and 5 pmol/L of probe (5´-FAM-CACTGTGATGTGTTCGAAGTTTGAGCA-TAMRA-3´). The cycler scheme consisted of 2 pre-PCR steps of 50°C for 15 min and 95°C for 2 min, followed by 45 cycles of 95°C for 15 s and 60°C for 30 s. Cycle threshold values were calculated by using PCR 7300 software (Applied Biosystems). FHV-1 nucleic acid was detected by PCR as described by Reubel et al.[12] and Stiles et al..[13]

Plasma samples were tested for feline leukemia virus (FeLV) antigen and antibodies against influenza virus A (H5N1), feline immunodeficiency virus (FIV), and feline coronavirus (FCoV). Antibodies to influenza virus were detected with a hemgglutination inhibition test according to the procedures of the World Organisation for Animal Health,[14] FeLV antigen was detected by using an ELISA (ViraCHEK/FeLV, Synbiotics Corporation, San Diego, CA, USA), and antibodies to FIV were detected by using an immunomigration test (Witness FIV, Synbiotics Corporation). Three dilutions (1:10, 1:100, and 1:400) of each plasma sample were tested for antibodies to group 1 coronaviruses by a modified indirect immunofluorescence assay.[15] Conjunctival, pharyngeal, and rectal swabs were cultured for pathologic bacterial infections.[16]

Two cats that seroconverted for H5N1 virus (cats 1 and 4) were humanely killed on day 50. Necropsy was performed on these 2 cats and on 12 other cats that had died during the observation period; organ homogenates (lung, liver, brain, trachea, tonsils, stomach, spleen, and pancreas) were tested for influenza virus-specific nucleic acids for each cat.


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