since the beginning of the 20th century , tiger populations have collapsed from an estimated 100,000 tigers among 9 subspecies to fewer than 3,500 individuals ( 1 , 2 ) , and all extant subspecies are currently listed as endangered or critically endangered by the international union for the conservation of nature ( http://www.iucn.org/ ) .
poaching , decimation of the tiger prey base , and habitat fragmentation have all contributed to tiger decline ( 1 , 3 , 4 ) . for the amur tiger ( panthera tigris altaica ) subspecies , 400 to 500 animals remain in the wild and represent one of the most endangered cat populations on the planet ( 2 ) .
usually reclusive and seldom observed , adult amur tigers have been seen uncharacteristically entering villages and wandering onto roadways in the russian far east ( rfe ) since 2001 . even more unusual is abnormal neurologic behavior in the tigers .
these observations , along with the diagnosis of morbillivirus infection in a wild amur tiger in 2004 ( histology and immunohistochemistry [ ihc ] findings were previously described ) , a cluster of wild tigers with abnormal neurologic behavior in 2010 , and a localized rapid and unexpected decline of tigers in the sikhote - alin biosphere reserve ( sabr ) in the rfe since 2009 ( 6 ) , led to concerns about an infectious disease and in particular canine distemper virus ( cdv ) as an emerging threat to amur tigers .
our aim was to identify the cause of neurologic disease and death in endangered amur tigers in the rfe , establish etiologic linkages between cases over a wide geographic and temporal range , and understand the significance of the disease in tiger conservation .
tissues collected during necropsy procedures from five adult , free - ranging tigers that died naturally or were destroyed in the rfe in 2001 , 2004 , or 2010 were available for histopathology , ihc staining , in situ hybridization ( ish ) , and reverse transcription - pcr ( rt - pcr ) testing .
brain tissue , critical in assessing cdv infection , was available from two tigers ( pt2004 and pt2010 - 3 ) ; lung , a primary site of cdv replication , was available from all tigers .
five to eighteen of twenty - two different tissue types ( adipose tissue , adrenal gland , artery , brain , heart , kidney , large intestine , liver , lung , lymph node , ovary , pancreas , peripheral nerve , salivary gland , skeletal muscle , spleen , stomach , small intestine , testis , tongue , trachea , or urinary bladder ) were available from each animal for histologic review .
bright - field microscopy was performed using a leica dm2500 microscope ( leica microsystems wetzlar gmbh , wetzlar , germany ) .
ihc for canine distemper virus antigen was performed using a primary monoclonal igg1 anti - cdv surface envelope antibody as described previously and included positive and negative controls ( 5 ) .
probes to a 600-bp nucleotide region of the phosphoprotein ( p ) gene of canine distemper virus were designed by panomics ( affymetrix , inc . ,
this region corresponds to nucleotides 1926 to 2526 of the cdv genome ( genbank accession no .
ish using fast red staining was performed using the panomics quantigene view rna kit for formalin - fixed paraffin - embedded sections according to the manufacturer s protocol ( product qv0050 , quantigene viewrna ffpe ; affymetrix , inc . ,
bright - field microscopy was performed as described above . for canine distemper virus rt - pcr
, rna was extracted using a standard protocol for xylene deparaffinization of formalin - fixed , paraffin - embedded tissue ( a total of 50  m [ 10 sections , 5  m each ] of tissue ; rneasy ffpe kit [ qiagen inc . , valencia ca ] ) .
primer sets were designed from the following regions of the cdv genome : p gene , morbf , 2132 to 2151 ; morbr , 2560 to 2541 ; cdvf4 , 2206 to 2228 ; and cdvr3 , 2319 to 2297 ; and hemagglutinin ( h ) gene , cdvh2-f , 8593 to 8619 ; cdvh2-r , 8842 to 8821 ; cdvh3-r , 8883 to 8864 ; cdvh4-r , 8868 to 8850 ; cdv - hf , 8521 to 8541 ; and cdv - hr , 8836 to 8815 .
one - step rt - pcr amplification of cdv p or h gene regions was performed using a standard protocol ( qiagen inc .
rt - pcr reactions were carried out using an initial 50c rt step for 30  min , using an annealing temperature of 45c for 45  s , and 40 cycles .
pcr products of correct molecular weight were purified using the exosap - it reagent ( affymetrix , santa clara , ca ) or the qiagen minelute gel extraction kit and directly sequenced in the forward and reverse directions using an abi 3730x dna analyzer for capillary electrophoresis and fluorescent dye terminator detection ( genewiz inc . , south plainfield , nj ) .
sequences were trimmed , aligned , and subjected to analysis using the blastn and blastx search tools to determine the identities of the viral sequences ( genbank , national center for biotechnology information ) .
the nucleotide sequences of the h gene were translated and aligned using the geneious alignment tool ( geneious pro 5.1.7 software ; biomatters ltd . , auckland , new zealand ) and analyzed for amino acid polymorphisms at positions 530 and 549 in the slam receptor binding region of the h gene . to determine the phylogenetic relationships of tiger cdvs to each other and to other cdv viruses and morbilliviruses , nucleotide sequences for the p and h genes from the tigers and representative cdv strains were aligned ( genbank , national center for biotechnology information ; http://www.ncbi.nlm.nih.gov ) ( geneious pro 5.1.7 software ; biomatters ltd . , auckland , new zealand ) .
pairwise identities were obtained by paup analysis to create a running p - distance pairwise comparison matrix ( paup plugin in geneious pro ) .
bayesian analysis was performed using mrbayes 3.1 plugin in geneious pro using gamma - distributed rate variation and an hky85 substitution model ( 8) .
the first 25% of a 1,100,000 chain length was discarded as burn - in , and 4 heated chains were run with a subsampling frequency of 200 .
trees were finalized and labeled ( figtree v1.3.1 software [ andrew rambaut , institute of evolutionary biology , university of edinburgh , 2006 to 2009 ; http://tree.bio.ed.ac.uk/ ] ) .
posterior probability values were calculated . between january and june 2010 , three adult free - ranging amur tigers ( panthera tigris altaica ) ( pt2010 - 1 , pt2010 - 2 , and pt2010 - 3 ) entered villages in the rfe ( fig . 
each was killed ( pt2010 - 1 and pt2010 - 3 ) or died naturally ( pt-2010 - 2 ) after exhibiting abnormal neurologic behavior ( disorientation , lack of response to stimulation , and/or nonaggressive fearlessness ) . prior to 2010
, two other free - ranging amur tigers ( pt2001 and pt2004 ) were captured and died after exhibiting similar neurologic behavior ( fig . 
four of the five tigers were emaciated or showed extreme weight loss at the time of death ( fig . 
1b ) . geographical distribution ( a ) and historical information ( b ) for tigers in the russian far east that died or were killed due to abnormal neurologic behavior in 2001 , 2004 , or 2010 .
( c ) tiger pt2010 - 3 : hematoxylin - and - eosin - stained section of brain with neuronal intranuclear eosinophilic viral inclusions ( arrow ) .
( d ) tiger pt2010 - 3 : positive immunohistochemical staining of neurons with monoclonal igg primary antibody to cdv viral envelope protein antigen ( arrows ) ( fast - red staining ) .
( e and f ) positive in situ hybridization ( fast red ) of probes to cdv p gene sequence in cdv - infected neurons in tiger pt2004 ( e ) and tiger pt2010 - 3 ( f ) .
brain tissue was available from pt2010 - 3 and pt2004 ( histology and ihc for the latter were previously reported , and tissue was reprocessed and reviewed for this article ) .
histologic lesions in the brains were identical and consisted of nonsuppurative viral encephalitis with severe demyelination .
brightly eosinophilic neuronal and glial cell nuclear viral inclusions and positive immunohistochemical staining in these cell types for an envelope component of cdv were also seen ( fig . 
the findings were severe and were sufficient to result in the clinically observed neurologic behavior in both cases and natural death in pt2004 ( 5 ) .
mild or moderate lymphoid depletion was seen in lymph nodes of pt2010 - 1 and pt2001 , respectively , and moderate lymphoid depletion was seen in spleens from pt-2001 , pt2004 , and pt2010 - 1 .
intralesional viral rna was confirmed in both tiger brains using ish to a 600-bp segment of the cdv p gene ( fig . 
viral inclusions , ihc staining , or ish consistent with cdv infection were not seen in nonneural tissues , including lung or lymphoid tissues , from any of the tigers ( data not shown ) .
extracted rna from select formalin - fixed paraffin - embedded ( ffpe ) tissues was analyzed by rt - pcr for morbillivirus and cdv phosphoprotein p and h genes using multiple primer sets .
positive results were obtained in 3 of 5 tigers : pt2004 ( histologic and ihc description previously reported ) , pt2010 - 2 , and pt-2010 - 3 .
cdv p gene products ranging in size from 114  bp to 430  bp and a 291-bp h gene product were recovered from the brains of both pt2004 and pt2010 - 3 . in pt2010 - 2 ,
lymph node tissue was positive for a 114-bp fragment of the cdv p gene and was h gene negative .
possible reasons for failure to recover h gene sequence from this tiger include rna degradation due to autolysis and/or cross - linking due to formalin fixation and/or prolonged formalin fixation prior to rt - pcr .
in addition to the possibility of true negatives , these complications could have prevented identification of positive cases among the remaining two tigers ( pt2001 and pt2010 - 1 ) or additional tissues in positive cases .
not having access to brain , the optimal target tissue in these neurologic tigers , may also explain the failure to identify additional positive tigers .
gene product sequences from pt2004 , pt2010 - 2 , and pt2010 - 3 were aligned with sequences of representative morbilliviruses , cdv sequences , and each other .
alignments of morbilliviruses and cdv strains were distributed as expected within viral clades and geographic distribution groups for asia , africa , europe , and north america .
h gene segments from pt2004 and pt2010 - 3 were 99.3% identical to each other ( fig . 
phylogenetic analysis segregated tiger h and p gene sequences within the arctic - like strains ( fig . 
blastn and paup distance matrix analysis showed tiger cdv h gene segments having closest identity ( 97.9% ) to arctic - like cdv strain 18133 ( 9 ) and a baikal seal ( phoca siberica ) strain ( 10 ) ( fig . 
our results indicate that tiger cdv is an arctic - like strain similar to those from greenland ( 11 ) , china ( 12 ) , russia ( 10 ) , and the united states ( 9 ) .
( a ) bayesian phylogenetic tree of the h gene nucleotide alignment from tigers pt2004 and pt2010 - 3 and representative cdv sequences obtained from genbank .
pairwise identities of nucleotide and amino acid sequences ( boldface ) between different strains of cdv were obtained through genbank and generated from a pairwise distance matrix calculated using paup software .
the critical amino acid residues g530 and y549 in the slam receptor binding domain of the cdv hemagglutinin protein have been shown to determine host cell tropism in vitro .
g  n , r , or d or y  h substitution at the 530 or 549 residue , respectively , is proposed to be associated with cdv transmission from domestic dogs ( canis lupus familiaris ) and disease emergence in novel host species ( 13 ) .
both tiger sequences lacked the y549  h substitution but contained an n residue at position g530 .
because the g530  n substitution is a consistent finding in arctic - like strains in general , including those in dogs and wildlife ( 1214 ) , we can not attribute a recent substitution event at this residue to disease emergence in tigers .
if this amino acid is under positive selective pressure , the change may have occurred through a dog - to - wildlife transmission prior to 1988 , when arctic - like strains , which include the g530  n mutation , were first detected in baikal seals and sled dogs in greenland ( 14 ) .
subsequent reintroduction of virus with this substitution into the domestic dog population may explain why the substitution is a predominant synapomorphy in the arctic - like lineage .
another interesting finding was three unique amino acid changes ( v538  i , t548  m , and d570  n ) in the tiger h gene sequence that have not been observed previously in arctic - like strains .
these findings suggest that the tiger arctic - like cdv is distinct ; however , additional information about arctic - like strains is needed to be confident in this conclusion .
cdv is the second most common cause of infectious disease death in domestic dogs and is a significant viral disease of global importance in common and endangered wild carnivores ( 15 ) .
it is a multihost pathogen , and interactions with and disease transmission from abundant wildlife reservoir species , such as raccoon dogs ( nyctereutes procyonoides ) or domestic dogs , are likely to be as important , if not more important , for disease transmission and population effect than infection among tigers alone due to low tiger numbers and population density ( 16 ) . in the rfe , little appears to be known about the distribution and strains of cdv that are circulating in domestic dogs and wildlife .
however , our identification of positive tiger cdv cases separated by 200  km to 300  km suggests wide distribution for the arctic - like cdv strain that infects and kills amur tigers .
low rates of vaccination and cdv infection are present in domestic dogs in russia , and direct transmission of cdv from infected , unvaccinated dogs to tigers is a significant concern , since amur tigers are known to encounter and kill domestic dogs ( 17 ) . in one survey , only 16% of village dogs were vaccinated against cdv and 58% of unvaccinated dogs were seropositive for antibodies to the virus , indicating high endemic exposure ( 18 ) . in the same report , 15% of wild tigers (
n = 40 ) sampled between 2000 and 2004 were seropositive for cdv antibodies , with no seropositive tigers detected prior to 2000 ( n = 27 ) ( 18 ) ; both pt2004 and pt2010 - 3 were seropositive for antibodies to cdv ( 1:256 ; virus neutralization [ vn ]  1:4 positive threshold value ) two ( 5 ) and three ( data not shown ) months , respectively , prior to their deaths .
cdv is a preventable infectious disease , and vaccination strategies , all of which have limitations and significant challenges in a wildlife setting ( 19 , 20 ) , are likely to be considered for protecting endangered amur tigers . because dogs are a known cdv reservoir , one strategy is to vaccinate domestic dogs to decrease transmission risk to susceptible wildlife .
this strategy was initiated in the serengeti ecosystem in 2003 in response to several significant cdv mortality events in lions ( panthera leo ) ( 21 ) .
the success of this strategy to date is unclear , since at least one cdv outbreak has occurred since initiation of the vaccination program ( 21 ) .
a second strategy is direct wildlife vaccination , which because of small numbers of animals , limited range , and known high disease - associated mortality is a critical component in conservation programs for the endangered black - footed ferret ( mustela nigripes ) ( 22 ) and critically endangered santa catalina island fox ( urocyon littoralis catalinae ) ( 23 ) .
vaccination with recombinant vectored vaccines has been safely used and is the recommendation for captive tigers in association of zoos and aquariums ( aza)-accredited zoos ( http://www.aazv.org/displaycommon.cfm?an=1&subarticlenbr=273 ) and for nondomestic canid and other wildlife species ( modified - live vaccines can induce disease and should not be used ) .
recombinant vectored vaccines may provide an option in wild tiger vaccination strategies , which in addition to safety must also consider efficacy , practicality , limitations , cost , and unintended consequences of vaccination ( including increased disease susceptibility to cdv or other pathogens ) in target or nontarget species ( 19 , 20 ) .
infectious disease as the cause of population decline or ( less commonly ) extinction in free - ranging wildlife is a recognized threat to species survival ; however , our ability to identify these events and their significance as they are occur and in time to mitigate their effects is rare ( 24 ) .
the absence of positive serology prior to 2000 ( 18 ) , lack of documented observations of neurologically ill tigers by scientists ( 5 , 6 , 18 ) or people living in tiger range ( personal communication , igor gregorivich ) prior to 2001 , and a cluster of cases in 2010 suggest cdv emergence after 2000 ( whether earlier individual cases or previous waves of tiger cdv infection and mortality occurred but were undetected prior to 2000 remains to be rigorously investigated ) .
additionally , in 2010 alone , cdv infection directly or indirectly killed approximately 1.0% of wild amur tigers ( 2 adults and 3 abandoned cubs ) .
these deaths reflect the immediate , direct effects of cdv infection and more than likely underestimate actual cdv - related deaths .
in addition and at the population level , the long - term impact of losing reproductively active animals , especially females like pt2010 - 3 , will exceed the direct effects of individual animal infection alone through lost productivity of both the dam and her offspring ( 25 ) .
our study is the first to confirm and genetically characterize a cdv that is killing wild , endangered amur tigers in the rfe .
our results indicate that tiger cdv is an arctic - like strain similar to cdv in baikal seals in russia and domestic dogs .
our report illustrates the importance of long - term wildlife monitoring and health surveillance in identifying emerging threats in endangered species .
it also shows how through these efforts we are afforded an opportunity to develop and implement mitigation activities , including identification of cdv reservoir species and consideration and assessment of vaccination strategies , to reduce disease risk in amur tigers and sympatric critically endangered amur leopards ( panthera pardus orientalis ) .
tiger cdv p and h gene sequences were deposited in genbank ( accession numbers kc579363 [ pt2004 ; h gene ] , kc579361 [ pt2004 ; p gene ] , and kc579362 [ pt2010 - 3 ; h gene ] ) .
accession numbers for tiger - derived sequences and all other sequences are presented in the figures .
tiger cdv p and h gene sequences were deposited in genbank ( accession numbers kc579363 [ pt2004 ; h gene ] , kc579361 [ pt2004 ; p gene ] , and kc579362 [ pt2010 - 3 ; h gene ] ) .
accession numbers for tiger - derived sequences and all other sequences are presented in the figures .
 abstractfewer than 500 amur tigers ( panthera tigris altaica ) remain in the wild . due to low numbers and their solitary and reclusive nature , tiger sightings across their range in the russian far east and china 
 are rare ; sightings of sick tigers are rarer still . serious neurologic disease observed in several wild tigers since 2001 suggested disease emergence in this endangered species . to investigate this possibility , histology , immunohistochemistry ( ihc ) , in situ hybridization ( ish ) , and reverse transcription - pcr ( rt - pcr ) 
 were performed on tissues from 5 affected tigers that died or were destroyed in 2001 , 2004 , or 2010 . 
 our results reveal canine distemper virus ( cdv ) infection as the cause of neurologic disease in two tigers and definitively establish infection in a third . 
 nonsuppurative encephalitis with demyelination , eosinophilic nuclear viral inclusions , and positive immunolabeling for cdv by ihc and ish were present in the two tigers with available brain tissue . 
 cdv phosphoprotein ( p ) and hemagglutinin ( h ) gene products were obtained from brains of these two tigers by rt - pcr , and a short fragment of cdv p gene sequence was detected in lymph node tissue of a third tiger . 
 phylogenetically , amur tiger cdv groups with an arctic - like strain in baikal seals ( phoca siberica ) . 
 our results , which include mapping the location of positive tigers and recognition of a cluster of cases in 2010 , coupled with a lack of reported cdv antibodies in amur tigers prior to 2000 suggest wide geographic distribution of cdv across the tiger range and recent emergence of cdv as a significant infectious disease threat to endangered amur tigers in the russian far east . 
Observation
Nucleotide sequence accession numbers.