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on march 25 , 2014 , health authorities reported that 43 of 84 goats on 5 breeding farms had died in huanan county in heilongjiang province after being transported from jiaxiang county in shandong province on march 13 .
animals showed clinical signs and symptoms of pprv infection , including fever ( temperature > 39.5c ) , ocular and nasal discharge , pneumonia , and severe diarrhea . on march 26
, health authorities reported that 13 of 87 imported breeding goats died of suspected ppr in huachuan county , which also borders huanan county .
these animals had also been transported from jiaxiang county on march 13 . because of this animal deaths ,
a systematic investigation of suspected ppr outbreaks was launched by the center for animal disease control and prevention of heilongjiang province .
after confirmation of the first case of ppr , active clinical surveillance was conducted by the local veterinary authorities , and farmers were urged to report suspected cases .
suspected flocks were defined as those that 1 ) during the previous month had 1 sheep or goats with 1 signs of illness : fever ( temperature > 39.5c ) , depression , anorexia , ocular and nasal discharge , pneumonia , necrosis and ulceration of mucous membranes , and severe diarrhea ; 2 ) had sheep or goats transported from other provinces , especially shandong province ; or 3 ) had neighboring farms on which animals had suspected pprv infections . during march 25may 5 ,
a total of 141 suspected flocks ( of which 41 contained imported animals [ defined as imported flocks ] ; other flocks were defined as local flocks ) from 13 counties were investigated .
sources of all introduced animals were livestock markets in shandong province , which were located mainly in 3 counties ( jiaxiang , heze , and liangshan ) . because spring is traditionally the time when farmers increase numbers of animals on their farms , breeding sheep or goats are bought from livestock markets for this purpose .
it takes 35 days for animals to be transported by truck from these markets to most destinations . from the 141 suspected flocks ,
1,887 serum samples were randomly collected from clinically healthy animals , and 285 nasal swab and 28 tissue samples ( lymph node , spleen , lung , and intestine ) were collected from all ill or dead animals .
competitive elisas were performed to detect antibodies against pprv in the serum samples as described ( 10,11 ) .
viral genomic rna was extracted from nasal swab and tissue samples by using the qiaamp viral rna mini kit ( qiagen , valencia , ca , usa ) according to the manufacturer s instructions .
pprv was detected by using a reverse transcription pcr ( rt - pcr ) specific for the 3 end of the nucleoprotein gene of pprv ; this rt - pcr yields an amplification product of 351 bp ( 12 ) .
pcr products were purified by using the qia quick gel extraction kit ( qiagen ) and sequenced .
multiple sequence alignment was performed by using clustalx2.0 ( 13 ) , and a phylogenetic tree was constructed by using the neighbor - joining method with mega6 ( 14 ) . a bootstrap analysis of 1,000 replicates was performed to test the degree of branching .
serologic analysis indicated that 17% ( 312/1887 ) of sampled animals and 76% ( 31/41 ) of imported flocks had antibodies against pprv ; all animals from suihua , yanshou , and qingan counties were antibody negative ( table ) .
a total of 29% ( 29/100 ) of local flocks in 5 counties ( zhaodong , hulan , huachuan , nenjiang , and baoqing ) had antibody - positive animals , which suggests that transmission of pprv between local and imported flocks had occurred in these areas . *
an antibody - positive flock contained 1 animal in positive for antibody against peste des petits ruminants virus .
a pcr - positive flock contained 1 animal positive for peste des petits ruminants virus by pcr .
rt - pcrs showed that 14% ( 39/285 ) of nasal swab samples and 89% ( 25/28 ) of tissue samples were positive for the nucleoprotein gene of pprv ; overall pprv positive rate was 20% ( 64/313 ) ( table ) .
combined results of serologic and the molecular diagnostic tests indicated that 43% ( 60/141 ) of suspected flocks were positive for pprv .
eleven outbreaks were confirmed in heilongjiang province ; the geographic distribution of these outbreaks is shown in figure 1 , panel b. to identify genetic characteristics of pprvs in heilongjiang province , a phylogenetic tree was generated by using the 3 ends of nucleoprotein genes of 49 viruses : 11 were obtained from rt - pcr  positive samples in this study and 38 were obtained from genbank .
seven of the 11 viruses belonged to lineage iv and were closely related to the strains tajikistan/04 ( 12 ) and tibet/07 ( 9 ) .
the remaining 4 viruses from this study belonged to lineage ii and were closely related to vaccine strain nigeria/75/1 ( figure 2 ) .
these 11 animals had not been vaccinated in shandong or heilongjiang provinces ; previous vaccination information was not available .
phylogenetic analysis of sequences of the 3 ends of nucleoprotein genes of peste des petits ruminant virus ( pprv ) , heilongjiang province , china , march 25may 5 , 2014 .
the tree was constructed by using the neighbor - joining method in mega6 ( 14 ) .
values along branches indicate bootstrap values of 1,000 replicates , and numbers on the right indicate lineages .
scale bar indicates estimated number of substitutions per 20 nt . to control and prevent further spread of ppr
these measures included 1 ) culling of all animals in pprv - infected flocks and on all farms within a radius of 5 km ; 2 ) restriction of transport of sheep and goats throughout the province ; 3 ) disinfection of pprv - contaminated areas and disposal of dead animal carcasses ; and 4 ) enhancement of the animal disease  reporting mechanisms . before the ppr outbreak , sheep and goats with signs and symptoms of disease
were not required to be reported to the local veterinary authorities , except for animals with foot - and - mouth disease .
after the outbreak , farmers were required to report all sick animals and vaccination of all susceptible animals .
the emergence of ppr in heilongjiang province poses a great threat to the livestock industry in this province .
our investigation showed that the most likely source of the outbreaks was animals from livestock markets in shandong province .
this finding suggests that transportation of unidentified pprv - infected sheep and goats is a major risk factor for spread of ppr in china .
although the partial nucleoprotein gene of the lineage ii viruses had similar sequence identity ( > 99% ) to that of vaccine strain 75/1 , we could not determine the vaccination status of these sick animals .
during march 25may 5 , 2014 , we investigated 11 outbreaks of peste des petits ruminants in heilongjiang province , china . 
 we found that the most likely source of the outbreaks was animals from livestock markets in shandong . 
 peste des petits ruminants viruses belonging to lineages ii and iv were detected in sick animals .