FULL PAPER Virology
An Outbreak of Canine Distemper Virus in Tigers (Panthera tigris): Possible
Transmission from Wild Animals to Zoo Animals
Yumiko NAGAO1), Yohei NISHIO1), Hiroshi SHIOMODA1), Seiji TAMARU1), Masayuki SHIMOJIMA1),
Megumi GOTO2), Yumi UNE3), Azusa SATO4), Yusuke IKEBE4) and Ken MAEDA1)*
1)Laboratory of Veterinary Microbiology, Faculty of Agriculture, Yamaguchi University, 1677–1 Yoshida, Yamaguchi 753–8515,
2)Laboratory of System Physiology, Faculty of Agriculture, Yamaguchi University, 1677–1 Yoshida, Yamaguchi 753–8515, Japan
3)Laboratory of Veterinary Pathology, Azabu University, 1–17–71 Fuchinobe, Kanagawa 252–5201, Japan
4)Akiyoshidai Safari Land, 1212 Aka, Mito, Mine, Yamaguchi 754–0302, Japan
(Received 10 November 2011/Accepted 16 December 2011/Published online in J-STAGE 28 December 2011)
ABSTRACT. Canine distemper virus (CDV), a morbillivirus that causes one of the most contagious and lethal viral diseases known in
canids, has an expanding host range, including wild animals. Since December 2009, several dead or dying wild raccoon dogs (Nyc-
tereutes procyonoides) were found in and around one safari-style zoo in Japan, and CDV was isolated from four of these animals. In
the subsequent months (January to February 2010), 12 tigers (Panthera tigris) in the zoo developed respiratory and gastrointestinal
diseases, and CDV RNA was detected in fecal samples of the examined tigers. In March 2010, one of the tigers developed a neuro-
logical disorder and died; CDV was isolated from the lung of this animal. Sequence analysis of the complete hemagglutinin (H) gene
and the signal peptide region of the fusion (F) gene showed high homology among these isolates (99.8–100%), indicating that CDV
might have been transmitted from raccoon dog to tiger. In addition, these isolates belonged to genotype Asia-1 and had lower homol-
ogy (<90%) to the vaccine strain (Onderstepoort). Seropositivity of lions (Panthera leo) in the zoo and wild bears (Ursus thibetanus)
captured around this area supported the theory that a CDV epidemic had occurred in many mammal species in and around the zoo.
These results indicate a risk of CDV transmission among many animal species, including large felids and endangered species.
KEY WORDS: canine distemper, raccoon dog, tiger.
doi: 10.1292/jvms.11-0509; J. Vet. Med. Sci. 74(6): 699–705, 2012
Canine distemper virus (CDV) is an enveloped, negative-
sense, single-stranded RNA virus  belonging to genus
Morbillivirus, family Paramyxoviridae, order Mononegavi-
rales, and causes lethal disease including pyrexia, anorexia,
nasal discharge, diarrhea, lymphopenia, and encephalitis
. Traditionally, CDV is well known to cause potentially
lethal disease among members of Canidae, Mustelidae,
and Procyonidae. Recently, CDV has been recognized as
a cause of morbidity and mortality in large felids, such
as lions (Panthera leo) in Tanzania’s Serengeti National
Park in 1994 ; lions, tigers (Panthera tigris), leopards
(Panthera pardus), and a jaguar (Panthera onca) in North
American zoos in 1991–1992 ; and one Siberian tiger
(Panthera tigris altaica) in Pokrovka, Russia, in 2004
. Furthermore, CDV infection also has been reported
in many non-carnivorous species, including collared pec-
caries (Tayassu tajacu) in Arizona  and non-human
primates (Macaca fuscata, Macaca fascicularis, Macaca
mulatta) [24, 30, 34, 39].
Lethal CDV infections in wild animals have occurred
worldwide. For instance, in the Serengeti, 30% of African
lions died of CDV infection ; and in California, the
number of island foxes (Urocyon littoralis catalane) de-
clined precipitously, with an approximate 95% reduction,
following CDV infection . In Japan, some epidemics of
CDV have been reported among wild animals. In Wakayama
Prefecture, 8 raccoon dogs (Nyctereutes procyonoides) and
one weasel (Mustela itatsi) died of CDV infection between
April 2007 and January 2008 . In Kochi Prefecture,
masked palm civets (Paguma larvata), Japanese badgers
(Meles meles), and a large number of raccoon dogs have
died of CDV infection since 2005 [37, 40].
In this study, we surveyed an apparent CDV epidemic in
wild raccoon dogs and zoo-housed tigers in and around one
safari-style zoo in Japan.
MATERIALS AND METHODS
Animals: Starting in December 2009, several wild
raccoon dogs were found dead or dying in and around a
safari-style zoo in Japan. From the end of January through
February 2010, 12 tigers in the zoo exhibited diarrhea,
vomiting, abdominal distress, and respiratory distress. Nine
out of twelve sick tigers eventually recovered, but one
of the tigers died with neuropathy on March 11, 2010. In
addition, two diseased tigers also died on January 21 and
February 5, 2010, although no post-mortem examination
*CorrespondenCe to: Maeda, K., Laboratory of Veterinary
Microbiology, Faculty of Agriculture, Yamaguchi University,
1677–1 Yoshida, Yamaguchi 753–8515, Japan.
©2012 The Japanese Society of Veterinary Science
Y. NAGAO ET AL.
Tissue samples: Tissue samples were collected for virus
isolation from four raccoon dogs and were analyzed to iden-
tify the causative agent. Fecal samples were collected from
seven diseased tigers, four males and three females (Table
1). Tissue samples from the dead tiger were collected for
virus isolation and histopathology.
Serum samples: Serum samples were collected from three
healthy lions in the zoo after the tiger infection episode, and
from nine wild bears that were captured from September 4
to October 29, 2010 in Yamaguchi Prefecture.
Cells: A72/cSLAM and CRFK/cSLAM cells expressing
canine signaling lymphocyte activation molecule (SLAM)
 were grown (at 37°C in 5% CO2) in Dulbecco’s modi-
fied Eagle’s medium (DMEM: GIBCO, Grand Island, NY,
U.S.A.) supplemented with 10% heat-inactivated fetal calf
serum (FCS, HyClone®, Ottawa, Canada), 100 U/ml peni-
cillin, and 100 µg/ml streptomycin (GIBCO).
Virus isolation: Swabs and fecal samples were mixed with
2 ml of DMEM containing antibiotics and then centrifuged
at 2,000 × g for 15 min at 4°C. The supernatants were filtered
through 0.45-µm filters (Millipore, Bedford, MA, U.S.A.).
Then, A72/cSLAM cells were inoculated with filtrates and
incubated until cytopathic effect (CPE) was observed.
Histopathology and immunostaining: Collected tissues
were fixed in 10% neutral buffered formalin and embedded
in paraffin wax. Sections (3 µm thick) were stained with
hematoxylin and eosin (HE). Separate paraffin sections were
examined by immunohistochemistry (IHC) using a polyclonal
antibody against CDV (Ikeda strain) . Secondary reac-
tions were performed with a peroxidase-conjugated Histofine-
Simple stain kit (Simplestain MAX-PO; Nichirei, Tokyo,
Japan), and visualized with 3,3’-diaminobenzidine and H2O2.
IHC slides were counterstained with Mayer’s hematoxylin.
For negative controls, the primary antibody was omitted.
Sequence analysis of hemagglutinin (H) gene: RNA was
extracted from virus-infected cells using QIAGEN RNA
Mini kit (QIAGEN, Hilden, Germany) and reverse-tran-
scription (RT) was carried out with random 9-mer primer
using TaKaRa RNA LA PCRTM kit (AMV) Ver.1.1 (Takara,
Shiga, Japan) at 30°C for 10 min, 42°C for 30 min, 70°C for
15 min and 5°C for 5 min. H gene was amplified using prim-
ers, CDV-HR (5’-AGA TGG ACC TCA GGG TAT AG-3’)
and CDV-HF (5’-AAC TTA GGG CTC AGG TAG TC-3’)
 at 94°C for 2 min and 30 cycles consisting of denatur-
ation at 94°C for 30 sec, annealing at 60°C for 30 sec, and
extension at 72°C for 3 min, followed by a final extension
at 72°C for 15 min. The amplified products were purified
by QIAquick PCR Purification kit (QIAGEN) and nucleo-
tide sequences were directly determined by ABI sequence
analyzer ABI 310 collection autosequencer (Applied Bio-
system, Carlsbad, CA, U.S.A.). For sequence analysis of H
gene, primers CDV-HF, CDV-HR, 204b (5’-GAA TTC GAT
TTC CGC GAT CTC C-3’), 232b- (5’-TAG GCA ACA CCA
ATA ATT TRG ACT C-3’) , 1F (5’-AGG TAT GTA CTA
TAG CAG TG-3’), 2F (5’-TAG TAA CCT GGA TGG TGC
CT-3’), 3F (5’-CCA GGG AAT CAA GTG GAA AT-3’), 4F
(5’-TCG AAC TCC AGT GAT GGC AA-3’), 1R (5’-AGG
CAC CAT CCA GGT TAC TA-3’), 2R (5’-CAC TGC TAT
AGT ACA TAC CT-3’), and 3R (5’TTT TGA CCC CAA
CTG CAT CG-3’) were used.
Sequence analysis of signal peptide region of fusion (F)
protein: To amplify the signal peptide region of F gene,
RT-PCR was performed using primers CDV-4713F (5’-
TCG CCT CTA GGA ATC TCA CT-3’) and CDV-5668R
(5’-GCA GTG ATT TGT GCA GCT GT-3’) at 94°C for 2
min and 30 cycles consisting of denaturation at 94°C for 30
sec, annealing at 60°C for 30 sec, and extension at 72°C for
1 min, followed by a final extension at 72°C for 15 min. The
amplified products were purified and then analyzed by direct
sequencing using primers CDV-4713F, CDV-5668R, 5275F
(5’-AAC TCA GGC TCT CAG TGC A-3’) and 5283R (5’-
TGC ACT GAG AGC CTG AGT T-3’).
Phylogenetic analysis: Homologies among our isolates
and other strains of CDV, including vaccine strains depos-
ited in Genbank, were analyzed using the GENETYX® Ver,
8. The evolutionary history was inferred using the neighbor-
joining method . Phylogenetic analyses were conducted
Detection of CDV genes in fecal samples: RNA was ex-
tracted from fecal samples of 7 tigers using QIAamp®Viral
RNA Mini Kit (QIAGEN) and RT was carried out as de-
scribed above. Genes encoding signal peptides of F protein
were amplified as described above and second PCR was
performed using TaKaRa Ex Taq (Takara) and primers
CDV-4713F and CDV-5283R as described above for the F
gene. Genes encoding partial nucleocapsid (N) protein were
amplified using primers, PP-I p1 (5’-ACA GGA TTG CTG
AGG ACC TAT-3’) and PP-I p2 (5’-CAA GAT AAC CAT
GTA CGG TGC-3’) at 94°C for 1 min and 40 cycles consist-
ing of denaturation at 94°C for 1 min, annealing at 59.5°C
for 2 min, and extension at 72°C for 1 min, followed by final
extension at 72°C for 5 min . PCR products were elec-
trophoresed on 2% agarose gel and stained with ethidium
bromide. This PCR for the N gene was reported to be very
sensitive to detect from clinical samples . Amplified
fragments were purified by QIAquick PCR Purification kit
(QIAGEN) to determine nucleotide sequences.
Detection of CDV in fecal samples collected from tigers
M♂ 5 years
a) N.D.; Not done, –; no band detected, +; rational size of band detected.
AN OUTBREAK OF CANINE DISTEMPER VIRUS IN TIGERS
Virus-neutralization (VN) test: VN test to KDK-1 
was carried out by 75% plaque-reduction neutralization test
(PRNT75) using our established cell line, CRFK/cSLAM
. In order to determine VN titers of the sera of lions
and bears, sera were diluted to 1:5 and then serially two-
fold diluted with DMEM containing 2% FCS. Diluted sera
were mixed with equal volumes of virus solution containing
100 plaque-forming units (PFUs) of KDK-1, followed by
incubation at 37°C for 1 hr. Then, 50 µl of mixtures were
added to each well of 24-well plates (Sumilon, Tokyo, Ja-
pan) containing subconfluent CRFK/cSLAM, and the plate
was incubated at 37°C for 1 hr, washed twice with DMEM
without FCS, and overlaid with DMEM containing 0.8%
agarose and 10% FCS. Plates then were incubated at 37°C
in 5% CO2 for 3–4 days. Cells were fixed with 5% buffered
formaldehyde for 1 hr and agarose layers were removed.
After staining with crystal violet, plaques were counted.
VN titer was expressed as the highest dilution of serum
that reduced plaques by more than 75% in comparison with
control wells without serum.
CDV epidemic in wild raccoon dogs and bred tigers: Since
December 2009, several wild raccoon dogs were found to be
dead or under anesthesia in and around one safari-style zoo
in Japan. Starting at the end of January 2010, 12 of 22 tigers
in the zoo exhibited diarrhea, vomiting, and respiratory
diseases. Two of the diseased tigers also died during this
outbreak, but tissue samples were not obtained from these
two tigers, nor was a cause of death determined. Another 10
of the affected tigers seemed to recover by the end of Febru-
ary, but one of the tigers developed a neurological disorder
and died on March 11, 2010.
Virus isolation from wild raccoon dogs and the tiger:
Three CDV strains, designated Yamaguchi/RD/091204,
Yamaguchi/RD/091207, and Yamaguchi/RD/091209, were
isolated from swabs of trachea of the raccoon dogs that died
on December 4, 7 and 9, 2009, respectively. A fourth CDV
strain, designated Yamaguchi/RD/091216, was isolated
from the brain of the raccoon dog that died on December
16, 2009. A fifth CDV strain (designated Yamaguchi/
WT/100311) was isolated from a lung of the tiger that died
on March 11, 2010. These viruses were confirmed as CDV
by RT-PCR and sequence analysis.
Histopathology in lung of the tiger: Histopathological
changes in the dead tiger were examined (Fig. 1). A diffuse
interstitial pneumonia with thick alveolar septa, marked hy-
perplasia of type II pneumocytes and many syncytial cells
were observed. A neutrophilic exudate was superimposed
on the proliferative alveolar lesion. In alveoli, there were
increased number of alveolar macrophages, edema, fibrin
exudation and necrotic epithelial cells and neutrophils
(Fig.1A). Most bronchiolar epithelium detached from bron-
chiolar wall and disappeared. Numerous eosinophilic inclu-
sion bodies mainly existed in alveolar epithelial syncytial
cells and CDV antigen was detected in alveolar epithelium
(Fig.1B). Inclusion bodies were also found within degenera-
tive intestinal epithelium cells.
Detection of CDV genes from fecal samples: During CDV
outbreak in tigers from February 11 to 22, fecal samples of
the seven tigers were collected at various days after onset of
diarrhea (Table 1). Samples were analyzed by RT-PCR for the
presence of CDV RNA, specifically a 245-bp segment of the
N gene and/or a 956-bp segment (via semi-nested RT-PCR)
of the signal peptide region of the F gene. The result showed
that CDV was detected from all examined tigers (Table 1).
The nucleotide sequences of the amplified products (245 bp)
were identical among fecal sample. The representative se-
quence of the N gene in this epidemic was deposited to DNA
Data Bank of Japan (DDBJ) for Yamaguchi/WT/100311
(Accession no. AB626080). To amplify the gene (956 bp)
containing the signal peptide region of F protein, RT-PCR
was carried out using primers CDV-4713F and CDV-5668R
for amplification. In the 1st PCR, CDV RNA was detected in
4 of 11 samples (4/7 tigers). The result of semi-nested PCR
using primers CDV-4713F and -5283R (571 bp) showed that
CDV was detected in 8 of 11 samples (6/7 tigers) (Table 1).
Sequence analysis of the signal peptide region (405 bp) of F
gene showed the sequence from one tiger ID S on February
14 was identical with those of Yamaguchi/WT/100311 (Ac-
cession no. AB619777), Yamaguchi/RD/091204 (Accession
no. AB619776) and Yamaguchi/RD/091216.
Nucleotide sequences of H genes: To analyze the homo-
Fig.1. Section of lung lesion containing eosinophilic inclusion bodies and syncytium. (A) Hematoxylin and
eosin (HE) staining. (B) IHC staining for CDV antigen.
Y. NAGAO ET AL.
genicity among strains isolated by cell culture, 2,022-bp
fragments containing the H gene were amplified from Yama-
guchi/RD/091204, Yamaguchi/RD/091216, and Yamaguchi/
WT/100311 by RT-PCR using primers CDV-HF and CDV-
HR, and the nucleotide sequences were determined by direct
sequencing method and deposited to DDBJ for Yamaguchi/
WT/100311 (Accession no. AB619774) and Yamaguchi/
RD/091204 (Accession no. AB619775). All H genes con-
sisted of 1824 bp encoding 607 amino acids. The predicted
H protein sequences of the two isolates from raccoon dogs
were identical, but the H gene of Yamaguchi/WT/100311
was predicted to encode a H protein with changes at amino
acid residues 165 and 272 (99.7% identity at the protein
level) (data not shown).
Comparison of H proteins among CDV strains: The
deduced amino acid sequences of H proteins were com-
pared. A substitution from tyrosine (Y) to histidine (H) at
the position of 549, which was speculated to be related to
CDV adaptation to wild carnivores , has been observed
in many Japanese isolates from wild animals , but this
Y549H substitution was not observed in CDV isolates
analyzed in the present study (Yamaguchi/WT/100311, Ya-
maguchi/RD/091204, and Yamaguchi/RD/091216). Amino
acid sequences of our isolates showed 95.9–98.4% identity
to those of other strains of genotype Asia-1, 91.8–94.2% to
those of other genotypes, and 89.5–89.9% identity to that of
Onderstepoort (data not shown).
Phylogenetic analysis of CDV isolates: Phylogenetic
analysis of the predicted H proteins was performed to iden-
tify the evolutionary relationship among these three CDV
isolates and other strains. All isolates in this CDV outbreak
formed a common cluster and were classified with the Asia-
1 genotype (Fig. 2).
Nucleotide sequences of the signal peptide region of F
proteins: Signal peptide region (amino acid residues 1–135)
of F protein is thought to be the most variable region in the
CDV genome . Therefore, nucleotide sequences of this
region were determined and deposited to DDBJ for Yama-
guchi/WT/100311 (Accession no. AB619777) and Yama-
guchi/RD/091204 (Accession no. AB619776). The results
revealed that the pre-peptide regions of our isolates were
completely identical to each other, but shared only 90.4–
94.3 and 82.2–90.4% identity (nucleotide and amino acid,
respectively) with the sequences of other Japanese strains
of genotype Asia-1. In particular, the identity with a major
vaccine strain (Onderstepoort) was as low as 80 and 62%
Fig. 2. Phylogenetic tree of CDV strains based on amino acid sequence of the hemagglutinin protein. Acces-
sion numbers of the sequences used are BAA19586 (Yanaka), BAA19584 (Ueno), BAA19585 (Hamamat-
su), BAA33740 (Tanu96), BAA84209 (KDK-1), BAK41514 (W729B), BAK41513 (W812B), CAA87688
(Danish Mink), CAA59358 (German Ferret), AAD18008 (A75/17), CAA90879 (Black Panther A92-6),
CAA87691 (American dog), CAA59359 (5804/Han90), AAM11476 (Dog Turkey), AAQ05829 (DK91 A),
CAB01252 (2544), BAB39167 (HM-3), BAB39166 (26D), BAA84208 (98-002), CAA87689 (Greenlandic
dog), CAA59357 (PDV-2), AAG15490 (SnyderHill), CAA84626 (Convac), AAK54669 (Onderstepoort).
The scale at the bottom indicates the units of the number of amino acid substitutions per site.
AN OUTBREAK OF CANINE DISTEMPER VIRUS IN TIGERS
(nucleotide and amino acid, respectively) (data not shown).
Seroprevalence of CDV in lions bred in the zoo and in
wild bears captured around the zoo: Sera from 3 lions
bred in the zoo were examined by VN test using KDK-1 as
described previously . By this VN test, we succeeded
in the detection of antibody to CDV in dogs, raccoons and
many wild animal species [15, 23, 26]. Only one of the 3
lions possessed VN antibody to CDV. The VN titer was high
at 1:320 (Table 2). Sera from 9 wild bears (Ursus thibeta-
nus) captured in this area after this CDV outbreak also were
examined by VN test; only one of the 9 samples possessed
VN antibody to CDV. The VN titer from this animal was
1:40 (Table 3).
Twelve tigers in the zoo exhibited diarrhea, vomiting,
abdominal distress, and respiratory distress starting at the
end of January, and one of the tigers died with neuropathy
on March 11, 2010. By IHC analysis and virus isolation,
CDV infection was etiologically involved in the death of
this tiger. Furthermore, CDV RNA was detected in at least
one sample from each animal between February 11 and 22,
indicating that a CDV outbreak occurred among tigers in
this zoo. On January 21 and February 5, another 2 of 12
diseased tigers at this site died after exhibiting diarrhea
and vomiting. Although no post-mortem examination was
performed, these tigers also were suspected to have been
killed by CDV infection. In this zoo, a total of 22 tigers were
housed in two different areas. Notably, 17 tigers that were
housed together (in one of these two areas) accounted for all
12 tigers exhibiting symptoms and all 3 deaths, indicating
that morbidity and mortality ratios of CDV in this group of
tigers were 71 and 25%, respectively.
Raccoon dogs are highly susceptible to CDV infection,
with high morbidity and mortality . CDV epidemics in
raccoon dogs, evidenced by animals exhibiting pneumonia
and gastroenteritis, have been reported previously in Japan
[18, 37, 40]. In our own previous study, we also observed
8 raccoon dogs dead by CDV infection in Tanabe City,
Wakayama Prefecture . In this study, several wild rac-
coon dogs, dead of apparent CDV infection, were discov-
ered around the zoo within a month before a CDV outbreak
among tigers housed at the zoo. Although the discovery and
diagnosis of dead and dying wild animals is a challenge,
raccoon dogs might be good indicators for CDV outbreaks
among wild animals because of this species’ high sensitivity
to CDV and high population density in Japan.
To analyze antigenic variation, phylogenetic analysis of
the H protein-encoding genes [5, 11, 22, 28] was performed
on several of the CDV isolates obtained in this study. The pre-
dicted H proteins revealed a very close relationship (99.7%
protein identity) among CDV from the raccoon dogs and the
dead tiger. Furthermore, our isolates from the raccoon dogs
and tiger were identical in the amino acid sequence even in
the signal peptide region of F protein, which is thought to
be most variable among isolates . These results indicate
that wild raccoon dogs and the tiger were infected with the
same CDV. Therefore, based on geographical and sequential
conditions, we conclude that this CDV epidemic occurred
by transmission of CDV from raccoon dogs (or other wild
animals around the zoo) to tigers. Although the exact routes
of transmission remain unknown, two possible routes are
suggested. One is that wild animals infected with CDV
entered into the zoo grounds and had direct contact with
the tigers, and another is that agents such as humans, food,
excreta, or so on played a role as a carrier. In this safari-style
zoo, the staffs carried out disinfection more carefully after
detection of CDV in raccoon dogs. However, the outbreak
occurred among tigers in this zoo. Therefore, we speculated
that CDV-infected wild animals must have become disorder
of central nervous system and invaded the zoo, even area
of tigers. From the ground and water contaminated by their
urine and feces containing CDV, tigers must have been in-
fected with CDV.
Our isolates did not have a substitution from tyrosine (Y)
to histidine (H) at hemagglutinin amino acid position 549,
although this substitution was speculated to be related to
CDV adaptation to wild carnivores . In addition, recent
isolates from Japanese wild animals possessed this substitu-
tion Y549H . Therefore, we propose that our isolates
might have originated from domestic dogs and might not
have been adapted well to wild carnivores. CDV transmis-
sion among different species has been reported previously.
In the case of Serengeti lions, domestic dog was considered
a likely source of CDV infection ; in California, wild
raccoons were hypothesized to transmit CDV to large cats
. In Japan, it was speculated that domestic dogs were the
sources of CDV infection of feral masked palm civets .
Although genotype Asia-1 was isolated previously from
dogs and wild animals in Japan [13, 14, 22], our results
demonstrate that this CDV genotype also can infect felids.
CDV seropositivity in one lion supports the CDV outbreak
in the zoo. In Japan, anti-CDV antibody was detected in 13
Table 2. Seroprevalence of CDV in lions in the zoo
Table 3. Seroprevalence of CDV in wild
bears captured in Yamaguchi Prefecture
Y. NAGAO ET AL.
of 20 lions at another zoo, although those animals exhibited
no obvious clinical sign of infection . In the Serengeti,
frequent interactions with jackals and spotted hyenas at kills
have been supposed to provide a potential mechanism for
transmission to lions . Moreover, seropositivity of one
wild bear (Ursus thibetanus) captured around the zoo sup-
ports the possibility of a CDV outbreak among wild mam-
mals around the zoo. As a predator of arctic seals, polar bear
(Ursus maritimus) has been suggested to be susceptible to
CDV infection . In the current study, it is assumed that
the wild bear might have been exposed by feeding on CDV-
infected wild animals. These seropositivities indicated that
many mammals in and around the zoo were exposed to CDV
during this episode.
In recent years, CDV seems to have broadened its host
range to include wild animals . All over the world, CDV
has led to mass mortalities in species ranging from wild ca-
nids (island fox, Urocyon littoralis) , felids (lion, Pan-
thera leo) , phocids (Bikal seal, Phoca siberica) ,
viverrids (masked palm civet, Paguma larvata) , and
procyonids (raccoon dogs, Nyctereutes procyonoides) [37,
40], to primates (crab-eating macaque, Macaca fascicularis
and rhesus monkeys, Macaca mulatta) [24, 30, 34]. The
increased host range of CDV suggests that this virus might
represent a possible zoonosis, as indicated by its detection
in non-human primates.
CDV epidemics also have threatened the continuation of
the small population such as island fox , giant panda
(Ailuropoda melanoleuca) , black-footed ferret (Mus-
tela nigripes)  and African wild dogs (Lycaon pictus)
. Thus the expanding host range of CDV represents a
challenge in the conservation of endangered animals. In the
Serengeti, since CDV vaccination to domestic dogs has been
adopted, CDV outbreak has not been observed in lions or
hyenas . Vaccines should be developed for wild and zoo
animals, especially endangered species; the CDV vaccine
used in dogs cannot be directly applied in other species,
because other animals exhibit different sensitivities to CDV
In conclusions, these results indicate a risk of CDV trans-
mission from wild animals to zoo animals including large
felids and endangered species.
ACKNOWLEDGMENTS. We thank Dr. Masami Mo-
chizuki (Kyoritsu Seiyaku Corporation) for kindly provid-
ing the KDK-1 strain. This study was supported by grants
from the Ministry of Education, Culture, Sports, Science
and Technology of Japan.
1. Appel, M. J. 1969. Pathogenesis of canine distemper. Am. J.
Vet. Res. 30: 1167–1182. [Medline]
2. Appel, M. J., Reggiardo, C., Summers, B. A., Pearce-Kelling,
S., Maré, C. J., Noon, T. H., Reed, R. E., Shively, J. N. and
Örvell, C. 1991. Canine distemper virus infection and en-
cephalitis in javelinas (collared peccaries). Arch. Virol. 119:
147–152. [Medline] [CrossRef]
3. Appel, M. J., Yates, R. A., Foley, G. L., Bernstein, J. J., Santi-
nelli, S., Spelman, L. H., Miller, L. D., Arp, L. H., Anderson,
M. and Barr, M. 1994. Canine distemper epizootic in lions,
tigers, and leopards in North America. J. Vet. Diagn. Invest. 6:
277–288. [Medline] [CrossRef]
4. Appel, M. J. and Summers, B. A. 1995. Pathogenicity of mor-
billiviruses for terrestrial carnivores. Vet. Microbiol. 44: 187–
191. [Medline] [CrossRef]
5. Blixenkrone-Möller, M., Svansson, V., Appel, M., Krogsrud,
P., Have, P. and Orvell, C. 1992. Antigenic relationship be-
tween field isolates of morbilliviruses from different carni-
vores. Arch. Virol. 123: 279–294. [Medline] [CrossRef]
6. Cattet, M. R., Duignan, P. J., House, C. A. and Aubin, D. J.
2004. Antibodies to canine distemper and phocine distemper
viruses in polar bears from the Canadian arctic. J. Wildl. Dis.
40: 338–342. [Medline]
7. Cleaveland, S., Appel, M. G., Chalmers, W. S., Chillingworth,
C., Kaare, M. and Dye, C. 2000. Serological and demographic
evidence for domestic dogs as a source of canine distemper
virus infection for Serengeti wildlife. Vet. Microbiol. 72: 217–
227. [Medline] [CrossRef]
8. Demeter, Z., Lakatos, B., Palade, E. A., Kozma, T., Forga’ch,
P. and Rusvai, M. 2007. Genetic diversity of Hungarian canine
distemper virus strains. Vet. Microbiol. 122: 258–269. [Med-
9. Endo, Y., Uema, M., Miura, R., Tsukiyama-Kohara, K., Tsuji-
moto, H., Yoneda, K. and Kai, C. 2004. Prevalence of canine
distemper virus, feline immunodeficiency virus and feline
leukemia virus in captive African lions (Panthera leo) in Ja-
pan. J. Vet. Med. Sci. 66: 1587–1589. [Medline] [CrossRef]
10. Frisk, A. L., Konig, M., Moritz, A. and Baumgartner, W. 1999.
Detection of Canine Distemper virus nucleoprotein RNA by
reverse transcription-PCR using serum, whole blood, and ce-
rebrospinal fluid from dogs with distemper. J. Clin. Microbiol.
37: 3634–3643. [Medline]
11. Haas, L., Martens, W., Greiser-Wilke, L., Mamaev, L., Butina,
T., Maack, D. and Barrett, T. 1997. Analysis of the hemagglu-
tinin gene of current wild-type canine distemper virus isolates
from Germany. Virus Res. 48: 165–171. [Medline] [CrossRef]
12. Harder, T. C. and Osterhaus, A. D. 1997. Canine distemper
virus-a morbillivirus in search of new hosts? Trends Micro-
biol. 5: 120–124. [Medline] [CrossRef]
13. Hirama, K., Goto, Y., Uema, M., Endo, Y., Miura, R. and Kai,
C. 2004. Phylogenetic analysis of the hemagglutinin (H) gene
of canine distemper viruses isolated from wild masked palm
civets (Paguma larvata). J. Vet. Med. Sci. 66: 1575–1578.
14. Iwatsuki, K., Miyashita, N., Yoshida, E., Gemma, T., Shin, Y.
S., Mori, T., Hirayama, N., Kai, C. and Mikami, T. 1997. Mo-
lecular and phylogenetic analyses of the haemagglutinin (H)
proteins of field isolates of canine distemper virus from natu-
rally infected dogs. J. Gen. Virol. 78: 373–380. [Medline]
15. Kameo, Y., Nagao, Y., Nishio, Y., Shimoda, H., Nakano, H.,
Suzuki, K., Une, Y., Sato, H., Shimojima, M. and Maeda, K.
2012. Epizootic canine distemper virus infection among wild
mammals. Vet. Microbiol. 154: 222–229. [Medline] [Cross-
16. Lamb, R. A. and Kolakofsky, D. 2001. Paramyxoviridae: the
viruses and their replication. pp. 1305–1443. In: Fields of Vi-
rology, 4th ed. (Knipe, D. M. and Howley, P. M. eds), Lippin-
cott Williams & Wilkins, Philadelphia.
17. Lee, M. S., Tsai, K. J., Chen, L. H., Chen, C. H., Liu, Y. P.,
Chang, C. C., Lee, S. H. and Hsu, W. L. 2010. The identifi-
AN OUTBREAK OF CANINE DISTEMPER VIRUS IN TIGERS Download full-text
cation of frequent variations in the fusion protein of canine
distemper virus. Vet. J. 183: 184–190. [Medline] [CrossRef]
18. Machida, N., Kiryu, K., Oh-ishi, K., Kanda, E., Izumisawa,
N. and Nakamura, T. 1993. Pathology and epidemiology of ca-
nine distemper in raccoon dogs (Nyctereutes procyonoides). J.
Comp. Pathol. 108: 383–392. [Medline] [CrossRef]
19. Mamaev, L. V., Visser, L. K., Belikov, S. I., Denikina, N.,
Harder, T., Goatley, L., Rima, B., Edginton, B., Osterhaus, A.
D. and Barrett, T. 1996. Canine distemper virus in Lake Bai-
kal seals (Phoca sibirica). Vet. Rec. 138: 437–439. [Medline]
20. Martella, V., Elia, G., Lucente, M. S., Decaro, N., Lorusso, E.,
Banyai, K., Blixenkrone-Moller, M., Lan, N. T., Yamaguchi,
R., Cirone, F., Carmichael, L. E. and Buonavoglia, C. 2007.
Genotyping canine distemper virus (CDV) by a hemi-nested
multiplex PCR provides a rapid approach for investigation
of CDV outbreaks. Vet. Microbiol. 122: 32–42. [Medline]
21. McCarthy, A. J., Shaw, M. A. and Goodman, S. J. 2007. Patho-
gen evolution and disease emergence in carnivores. Proc. Biol.
Sci. 274: 3165–3174. [Medline] [CrossRef]
22. Mochizuki, M., Hashimoto, M., Hagiwara, S., Yoshida, Y.
and Ishiguro, S. 1999. Genotypes of canine distemper virus
determined by analysis of the hemagglutinin genes of recent
isolates from dogs in Japan. J. Clin. Microbiol. 37: 2936–2942.
23. Mochizuki, M., Motoyoshi, M., Maeda, K. and Kai, K. 2002.
Complement-mediated neutralization of canine distemper vi-
rus in vitro: cross-reaction between vaccine Onderstepoort
and field KDK-1 strains with different hemagglutinin gene
characteristics. Clin. Diagn. Lab. Immunol. 9: 921–924. [Med-
24. Morikawa, S. 2008. Outbreak of deadly canine distemper vi-
rus infection among imported cynomolgus (crab-eating) mon-
keys. Infect. Agents Surveill. Rep. 29: 315.
25. Nakano, H., Kameo, Y., Andoh, K., Ohno, Y., Mochizuki, M.
and Maeda, K. 2009. Establishment of canine and feline cells
expressing canine signaling lymphocyte activation molecule
for canine distemper virus study. Vet. Microbiol. 133: 179–
183. [Medline] [CrossRef]
26. Nakano, H., Kameo, Y., Sato, H., Mochizuki, M., Yokoyama,
M., Uni, S., Shibasaki, T. and Maeda, K. 2009. Detection of
antibody to canine distemper virus in wild raccoons (Procyon
lotor) in Japan. J. Vet. Med. Sci. 71: 1661–1663. [Medline]
27. Nunoya, T., Tajima, M., Ishikawa, Y., Samejima, T., Ishikawa,
H. and Hasegawa, K. 1990. Occurrence of a canine distemper-
like disease in aquarium seals. Nippon Juigaku Zasshi 52:
469–477. [Medline] [CrossRef]
28. Orvell, C., Bilxenkrone-Moller, M., Svansson, V. and Have, P.
1990. Immunological relationships between phocid and canine
distemper virus studied with monoclonal antibodies. J. Gen.
Virol. 71: 2085–2092. [Medline] [CrossRef]
29. Qin, Q., Li, D., Zhang, H., Hou, R., Zhang, Z., Zhang, C.,
Zhang, J. and Wei, F. 2010. Serosurvey of selected viruses in
captive giant pandas (Ailuropoda melanoleuca) in China. Vet.
Microbiol. 142: 199–204. [Medline] [CrossRef]
30. Qiu, W., Zheng, Y., Zhang, S., Fan, Q., Liu, H., Zhang, F.,
Wang, W., Liao, G. and Hu, R. 2011. Canine distemper out-
break in rhesus monkeys, China. Emerg. Infect. Dis. 17: 1541–
31. Quigley, K. S., Evermann, J. F., Leathers, C. W., Armstrong,
D. L., Goodrich, J., Duncan, N. M. and Miquelle, D. G. 2010.
Morbillivirus infection on a wild Siberian tiger in the Russian
Far East. J. Wildl. Dis. 46: 1252–1256. [Medline]
32. Roelke-Parker, M. E., Munson, L., Packer, C., Kock, R.,
Cleaveland, S., Carpenter, M., O‘Brien, S. J., Pospischil, A.,
Hofmann-Lehmann, R. and Lutz, H. 1996. A canine distemper
virus epidemic in Serengeti lions (Panthera leo). Nature 379:
441–445. [Medline] [CrossRef]
33. Saitou, N. and Nei, M. 1987. The neighbor-joining method: a
new method for reconstructing phylogenetic trees. Mol. Biol.
Evol. 4: 406–425. [Medline]
34. Sun, Z., Li, A., Ye, H., Shi, Y., Hu, Z. and Zeng, L. 2010. Natu-
ral infection with canine distemper virus in hand-feeding Rhe-
sus monkeys in China. Vet. Microbiol. 141: 374–378. [Med-
35. Timm, S. F., Munson, L., Summers, B. A., Terio, K. A., Dubo-
vi, E. J., Rupprecht, C. E., Kapil, S. and Garcelon, D. K. 2009.
A suspected canine distemper epidemic as the cause of a cata-
strophic decline in Santa Catalina Island foxes (Urocyon lit-
toralis catalinae). J. Wildl. Dis. 45: 333–343. [Medline]
36. van de Bildt, M. W., Kuiken, T., Visee, A. M., Lema, S., Fitz-
john, T. R. and Osterhaus, A. D. 2002. Distemper outbreak
and its effect on African wild dog conservation. Emerg. Infect.
Dis. 8: 211–213. [Medline] [CrossRef]
37. Watabe, T. and Yoshizawa, M. 2006. The outbreak of death
frequent occurrence of the wild raccoon dog by canine distem-
per. J. Environ. Dis. 15: 11–14 (in Japanese).
38. Williams, E. S. and Thorne, E. T. 1996. Infectious and para-
sitic diseases of captive carnivores, with special emphasis on
the black-footed ferret (Mustela nigripes). Rev. Sci. Tech. 15:
91–114. [Medline] [CrossRef]
39. Yoshikawa, Y., Ochikubo, F., Matsubara, Y., Tsuruoka, H.,
Ishii, M., Shirota, K., Nomura, Y., Sugiyama, M. and Yama-
nouchi, K. 1989. Natural infection with canine distemper virus
in a Japanese monkey (Macaca fuscata). Vet. Microbiol. 20:
193–205. [Medline] [CrossRef]
40. Yoshizawa, M. and Watabe, T. 2007. The canine distemper
outbreak situation of mammals in Kochi City and the out-
skirts. Kagawa Seibutsu 34: 63–67 (in Japanese).