Naturally occurring influenza infection in a ferret
(Mustela putorius furo) colony
Abby R. Patterson,1Vickie L. Cooper, Kyoung-Jin Yoon, Bruce H. Janke, Phillip C. Gauger
an outbreak of respiratory disease were submitted to the Iowa State University Veterinary Diagnostic
Laboratory. Microscopic examination of lung samples revealed bronchointerstitial pneumonia with
necrotizing bronchiolitis. Influenza A virus was detected in sections of formalin-fixed lung by immunohisto-
chemistry and reverse transcription polymerase chain reaction assay. A field investigation of the premises and
analysis of additional samples led to the confirmation and characterization of an influenza virus with high
homology to contemporary reassortant H1N1 swine influenza viruses. Although ferrets have been used
extensively to research the virulence and transmissibility of avian, human, and swine influenza virus strains, no
published information exists on naturally occurring outbreaks of swine influenza in ferrets.
Tissue samples from 2 juvenile ferrets (Mustela putorius furo) from a colony that was undergoing
Key words:Influenza virus; Mustela putorius furo; natural infection; Swine influenza virus.
A routine diagnostic case that consisted of pooled tissue
samples from 2 juvenile ferrets (Mustela putorius furo) with
a history of clinical respiratory disease was submitted to the
Iowa State University Veterinary Diagnostic Laboratory
(Ames, IA). The ferrets represented a population of
approximately 1,000, of which 8% of the animals were
exhibiting respiratory signs, including severe dyspnea,
sneezing, coughing, and crusting of the eyes and nose.
Mortality at the time of the first submission was 0.6%.
Field investigation of the farm undertaken 2.5 weeks after
the initial submission revealed the presence of multiple
avian species (including ducks, geese, and peacocks), small
mammal species raised for purchase (raccoons, skunks, and
fox), a cow and calf herd, horses, and llamas. The ferrets
were housed in individual cages constructed with wood and
galvanized wire. Ferrets were hand fed a ground turkey and
chicken mixture and hand watered daily. The farm was not
routinely tested for pathogens; however, 4 years before the
above-described case, the farm experienced similar clinical
signs. In both cases, clinical signs occurred in early spring.
Clinical signs among the ferrets were still apparent, and
additional samples were collected to characterize the
influenza virus strain that was affecting the colony. Nasal
swabs, bronchoalveolar lavage samples, serum, and tissues
were collected from 4 ferrets that displayed acute clinical
signs. Serum samples from 4 ferrets that were not showing
acute clinical signs were also obtained for serologic
Microscopic examination of lung sections revealed
bronchointerstitial pneumonia with necrosuppurative
bronchiolitis. This was characterized by patchy areas of
alveolar septal thickening; pulmonary congestion; coagula
of debris in alveoli, with associated atelectasis; and a locally
extensive region in which airways were dilated and filled
with neutrophils, macrophages, degenerate cells, and
debris. Scattered airways were characterized by attenuation
of airway epithelium, bronchiolitis, and bronchiogenic
abscessation (Fig. 1). No bacterial or mycoplasmal agents
were isolated from the lung tissue. Canine distemper virus
(CDV) was not detected by fluorescent antibody test5or
real-time reverse transcription polymerase chain reaction
(real-time RT-PCR) by using a set of primers and probe
targeted to the nucleoprotein gene of CDV.19Influenza A
virus (family Orthomyxoviridae, genus Influenzavirus A;
H1N1 subtype) was detected in the lung tissue by
immunohistochemistry22(IHC; Fig. 2) and by real-time
RT-PCR.11Briefly, the IHC procedure used a monoclonal
antibody, which targeted the nucleoprotein of Swine
influenza virus (SIV),aa biotinylated goat anti-mouse
antibody,bperoxidase-conjugated streptavidin,band di-
aminobenzidine tetrahydrochloride solution.cThe primers
hemagglutinin (H1 and H3),and neuraminidase (N1 andN2)
gene segments of the SIV genome.
The sequence analysis of the hemagglutinin (HA) gene
(GenBank accession no. FJ50522) indicated high homology
(98%) with that of viruses that belong to one of the major
clades of triple reassortant H1N1 SIV represented by
MN02 SwH1N1 (Fig. 3). Influenza A virus, which was
subtyped H1N1, was isolated from 3 of 4 lung sample
homogenates collected during the follow-up visit by using
both the Madin–Darby canine kidney cell line and
embryonated chicken eggs.23,25There was a 99.7% nucle-
From the Department of Veterinary Diagnostic and Production
Animal Medicine, College of Veterinary Medicine, Iowa State
University, Ames, IA.
1Corresponding Author: Abby R. Patterson, 1653 Veterinary
Medicine Building, 1600 South 16th Street, Ames, IA 50010.
J Vet Diagn Invest 21:527–530 (2009)
otide homology for the HA gene between the viruses
recovered from the first and second submissions (Fig. 3).
The sequence analysis for the matrix gene (GenBank
accession no. GQ149509) of the isolates revealed that the
virus was from a swine lineage in origin (Fig. 4).
Serum samples from the ferrets were tested by hemag-
glutination inhibition (HI) assay3against a variant swine
reassortant H1N1 (SIV99), a classic swine H1N1 (IA76), a
cluster I H3N2 (TX98) SIV, and the ferret H1N1 isolate.
Inhibition titers exhibited high titers (particularly in
convalescent sera) to the ferret isolate, moderate titers to
the variant swine reassortant and the cluster I H3N2
isolate, and low titers to the classic swine H1N1 isolate
(Table 1). Titers to the cluster I H3N2 isolate could be
nonspecific or indicative of previous exposure to H3 virus
of human, swine, equine, or canine. The HA gene of TX98
is human lineage in origin. A low degree of cross reactivity
among swine H3, equine H3, and canine H3 can be
Because of similarities in the clinical signs, pathogenesis
of disease, and immunologic response induced in both
humans and ferrets,15the ferret model has been extensively
used to assess the pathogenicity of avian, swine, and human
Influenza A virus isolates.2,9,14,26Clinical signs and lesions
induced by swine, avian, and human Influenza A virus
isolates in ferrets have been well characterized in previous
studies and were similar to those observed in the naturally
infected animals in the current study.12,13,15Natural
illustrating the presence of influenza virus antigen within
bronchiolar epithelial cells. Bar 5 100 mm.
Immunohistochemical stain of the ferret lung,
ferret infected with Influenza A virus. Hematoxylin and eosin. Bar
5 200 mm.
Necrosuppurative bronchiolitis in the lung of a
(Hu) influenza viruses and the influenza virus in lung tissue from a clinically ill ferret (08-13304) based on nucleotide sequence of the
hemagglutinin RNA segment. Reliability of the tree was assessed by bootstrap analysis with 1,000 replications. The GenBank accession
number of each reference virus is given within the parentheses.
Phylogenetic relationship of ferret isolate (08-15828) of influenza A virus to representative H1 swine (Sw) and human
infection of a ferret colony with a human influenza virus
was reported in 19481; however, to the authors’ knowledge,
natural infections in colonies with a swine isolate have not
previously been reported.
The specific source of infection was not identified in the
present case, but the close association with other birds and
mammals offered several possible sources of transmission.
No swine were housed on the farm, but a possible source of
infection was aerosol transmission from a swine operation
located approximately 0.25 miles from the ferret colony.
Influenza viruses are nearly ubiquitous in swine,14and it is
not uncommon for swine operations to be endemically
infected.17In addition, aerosol transmission between
adjacent swine operations was reported as a potential
means of transmission.6The fact that the virus isolated
from the ferret colony had an HA gene with 98% nucleotide
homology to that of a SIV isolate (MN02 SwH1N1) and
also contained the matrix gene of SIV origin suggests likely
transmission from the adjacent swine operation. Unfortu-
ferrets (A–D) and previously exposed but clinically normal
ferrets (E–H) by using a variant H1N1 (SIV99), a classic H1N1
(IA76), a cluster I H3N2 (TX98) SIV, or the virus isolated from
the affected ferrets (Ferret H1N1).*
Hemagglutination titers from acutely affected
Ferret SIV99IA76TX98 Ferret H1N1
* SIV 5 Swine influenza virus.
avian (Av) influenza viruses based on nucleotide sequence of the matrix RNA segment. Phylogenetic and molecular evolutionary
analyses were conducted by using MEGA version 4. The tree was generated by using the neighbor-joining method. Reliability of the tree
was assessed by bootstrap analysis with 1,000 replications. GenBank accession numbers are in parentheses.
Phylogenetic relationship of ferret isolate (08-15828) of Influenza A virus to representative swine (Sw), human (Hu), and
Case Reports 529
nately, because of the inability to test surrounding swine Download full-text
operations, this hypothesis could not be confirmed. No
evidence of horizontal transmission through uncooked
swine byproducts, as previously reported in a mink
population,8was identified in this colony. However,
uncooked turkey was included in the diet. This poses a
risk, because turkeys are known to carry SIVs.4Because
numerous species, such as horses,24dogs,18minks,7cats,16
raccoons,10and marine mammals,21can act as potential
reservoirs for maintenance and transmission to natural
hosts, the potential for horizontal transmission from one of
the other species on the farm cannot be ruled out.
Another possible source of infection would include
interaction with infected human workers that results in
transmission of a swine H1N1 strain to the colony. The
ability of the ferret to transmit influenza virus to humans
was reported in the 1930s.20Subsequent studies confirmed
the infectious nature of human influenza isolates in ferrets.
However, workers on the farm with the ferret colony had
no known contact with swine, and influenza was not
diagnosed by a physician in any of the workers at the time
of the outbreak.
Although the specific source of infection was not
identified, based on nucleotide sequence and HI analysis,
the most likely source of infection was from an adjacent
swine operation. To the author’s knowledge, the present
case represents the first report of a natural swine influenza
infection in a ferret colony. In the course of the
investigation, the colony was treated with injectable
antibiotics to prevent secondary bacterial infections. The
outbreak ended without further intervention; vaccination
of the herd was discussed but not implemented.
The authors would like to thank Joe
Bender, Amy Chriswell, Fanghong Zhou, and Suzanne Block
submission; and Pfizer Animal Health for financial support.
Sources and manufacturers
a. VMRI, Ames, IA, not commercially available.
b. DAKOH Labeled Streptavidin-Biotin 2 System kit, Dako
North America Inc., Carpinteria, CA.
c. DAKOH Liquid DAB (diaminobenzidine) Substrate-Chromo-
gen System, Dako North America Inc., Carpinteria, CA.
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