study sites : this study was conducted in four counties ( gangcha , haiyan , 
 xinghai and chengduo counties ) in northeastern and southern qinghai province , china ( fig . 1fig .
bold lines represent 
 district borders , and thin lines represent county borders . 1 : gangcha county , 2 : 
 haiyan county , 3 : xinghai county ( heka town ) , 4 : chengduo county . * : xining 
 ( provincial capital ) . ) .
all of the study sites were inhabited by nomadic tibetans and other minority ethnic 
 groups grazing yak and sheep on the vast grasslands of the qinghai - tibetan plateau .
the 
 altitudes of the study sites ranged from 3,000 to 4,000 m. study sites .
bold lines represent 
 district borders , and thin lines represent county borders . 1 : gangcha county , 2 : 
 haiyan county , 3 : xinghai county ( heka town ) , 4 : chengduo county . * : xining 
 ( provincial capital ) . collection of dog feces : to collect dog feces , we first asked local 
 farmers to chain their dogs to prevent them from running away to ensure that they would 
 defecate nearby . at heka town in xinghai county ,
dog feces were collected before 
 administration of 5 to 10 mg / kg praziquantel and then at 24 and 48 hr thereafter to 
 determine the optimal time for detecting eggs from feces by fecal egg examination . upon 
 finding tapeworms in feces after deworming ,
they were collected and identified to species by 
 dna barcoding . except for chengduo county where deworming was not performed ,
dog feces were 
 collected at 24 hr after praziquantel administration in all study sites .
all feces samples 
 were frozen at 80c for at least 10 days to render eggs uninfective . fecal egg examination : fecal egg examination was performed by the 
 centrifugal sucrose flotation technique using 1.0 g of feces . upon observing taeniid eggs , 2 
 to 40 eggs were collected from each feces sample under a stereomicroscope , and the eggs were 
 identified to species by dna barcoding .
molecular identification of parasite species ( dna barcoding ) : dna was 
 extracted from adult cestodes or eggs using a qiaamp dna mini kit ( qiagen , tokyo , japan ) 
 following the manufacturer s instructions .
as reported previously , partial segments of the parasite mitochondrial cytochrome 
 c oxidase subunit 1 ( cox1 ) and nadh dehydrogenase 
 ( nad1 ) gene were amplified by polymerase chain reaction ( pcr ) using 
 primers 2575 ( 5-ttt ttt ggg cat cct gag gtt tat-3 ) and 3021 ( 5-taa aga aag aac ata atg 
 aaa atg-3 )   for cox1 and nad1t - fw 
 ( 5-ggk tat tct car ttt cgt aag gg-3 ) and nad1t - rv ( 5-atc aaa tgg agt acg att agt ytc 
 ac-3 )   for nad1 .
the amplified 
 products ( 443 and 507 base pairs for cox1 and nad1 , 
 respectively ) were examined by agarose gel electrophoresis , and their nucleotide sequences 
 were determined as described previously .
the 
 obtained sequences were aligned using clc sequence viewer 6.1 ( clc bio japan , tokyo , japan ) , 
 and a basic local alignment search tool ( blast ) similarity search was conducted for species 
 and genotype identification .
deworming of dogs : a control trial to assess taeniid infection in farm 
 dogs was conducted in the town of heka in xinghai county where the prevalence of taeniid 
 cestodes in dogs was the highest .
a total of 90 dogs were selected for the trial . with 
 support of a local veterinarian ,
dog owners were requested to administer the required dose 
 of praziquantel ( 510 mg / kg ) to each dog at 45-day intervals from july 2010 for two years . 

change in the prevalence of infection with taeniid cestodes was then assessed by comparing 
 the prevalence before the beginning of the trial and those at 1 and 2 years after starting 
 the trial .
in addition , in order to confirm the efficacy of praziquantel treatment , feces 
 samples were collected and examined again from all dogs 1 month after the 2nd year 
 examination when all dogs were dewormed properly for sampling feces . questionnaire survey : since the disparity in taeniid cestode prevalence in 
 dogs was greatest between the counties of xinghai and haiyan , a questionnaire survey was 
 conducted among 30 farmers in these 2 counties to assess their knowledge of echinococcosis 
 and their feeding and contact with their dogs .
statistical analysis : difference in the questionnaire responses between 
 the 2 counties was evaluated by fisher s exact test implemented in the program r .
fecal egg examination : the results of fecal egg examination performed on 
 feces collected from 43 dogs before praziquantel administration and then at 24 and 48 hr 
 thereafter are shown in table 1table 1.comparison of taeniid egg detection rate by fecal examination in feces samples 
 collected before and after deworming dogs and the number of dogs that excreted 
 cestodes after dewormingtaeniid speciesno
dogsexcreting cestodestiming of samplingbeforeafter 24 hrafter 48 hrtaenia hydatigena18817611taenia multiceps11111none240000total439 ( 21%)18 ( 43%)*7 ( 17%)*12 ( 29% ) * * : because an owner refused to have his dog given anthelmintic , a total number 
 examined was 42 .. taeniid eggs were found in 9 ( 21% ) , 18 ( 43% ) and 7 ( 17% ) feces samples , 
 respectively , indicating detection rate was highest in feces collected at 24 hr after 
 deworming .
dna barcoding of the excreted eggs and adult cestodes revealed 
 that 18 dogs were infected with t. hydatigena and one dog with t. 
 multiceps .
eggs of other parasite species were not found . * : because an owner refused to have his dog given anthelmintic , a total number 
 examined was 42 .
prevalence of taeniid cestodes in dogs at four different study sites : the 
 prevalence of taeniid eggs in the feces of dogs from 4 counties examined in 2010 is shown in 
 table 2table 2.prevalence of taeniid cestodes determined by fecal egg examination in dogs from 
 four counties in qinghai province , chinacountyno .
positivetaeniid speciesechinococcus granulosustaenia hydatigenataenia multicepshaiyan380nananagangcha77141121xinghai903112181chengduo729630total2775419332na : not applicable . * a ) fecal egg examination with deworming .
* b ) fecal egg 
 examination without deworming .. the highest prevalence was found in xinghai county , and no positive samples 
 were found in haiyan county .
dna barcoding of egg dna showed that e. 
 granulosus and t. hydatigena were detected in 3 of the counties 
 and that xinghai county had the highest prevalence of both parasite species .
all 
 nad1 sequences of e. granulosus were identical to that 
 registered in genbank as g1 genotype ( accession no .
in addition to taeniid eggs , trichuris vulpis eggs were found in 1 dog from 
 gangcha county and in 2 dogs from xinghai county .
control trial of taeniid infection in dogs : although the trial was started 
 with 90 dogs , the number of animals decreased to 41 over the course of the trial due to 
 death of dogs , transfer of dogs , not having of dogs chained at the time of sampling , 
 disappearance of dogs and owners and owners rejecting further deworming treatments because 
 of changes in dog health after dosing ( table 
 3table 3.change in taeniid cestode prevalence in dogs over the course of a 2-year 
 anthelmintic control trial in xinghai county , chinataeniid speciespre - trialone year post - trialtwo years post - trialno .
the overall prevalence of taeniid cestodes was reduced from 34.4% at the 
 beginning of the trial to 9.6% and to 4.9% 1 and 2 years after starting the trial , 
 respectively .
for 
 example , e. granulosus prevalence changed from 13.3% to 3.6% and to 2.4% , 
 t. hydatigena prevalence changed from 20.0% to 2.4% and to 2.4% , and 
 t. multiceps prevalence changed from 1.1% to 3.6% and to 0% 1 and 2 years 
 after starting the trial , respectively .
only 2 dogs were positive for taeniid eggs in 
 consecutive years of the study , but both dogs were infected by different taeniid species in 
 different years ; 1 dog was infected with t. hydatigena before anthelmintic 
 treatment , but with e. granulosus at the end of the first year , and the 
 other dog was infected with e. granulosus at the end of the 1st year , but 
 with t. hydatigena at the end of the 2nd year .
when dogs were examined for 
 the last time , i.e. 1 month after the 2nd year examination , no eggs were detected in any of 
 the dog feces .
farmers awareness of echinococcosis and animal management : the responses 
 to the questionnaire are shown in table 
 4table 4.comparison of questionnaire responses from farmers in haiyan and xinghai 
 counties 
 . although obvious differences were not observed in the feeding behavior of the 
 respective dogs or contact frequency with their dogs , significant difference was observed in 
 the awareness of echinococcosis and the knowledge of transmission risk and lifecycle .
infection by e. granulosus and t. hydatigena in dogs was 
 detected in 3 of the 4 counties , indicating the species are distributed widely in qinghai 
 province .
these findings are consistent with reports of cystic echinococcosis in humans and 
 domestic animals elsewhere on the qinghai - tibetan plateau [ 5 , 9 , 13 , 14 , 26 , 29,30,31 ] . finding t. 
 hydatigena metacestodes at abattoirs seems to be very common , and
the prevalence 
 was reported to be 8.230.8% in sheep [ 11 , 20 ] . while , t. multiceps was only found 
 in 2 dogs , and the prevalence of this parasite in qinghai province is moderate and
has been 
 reported in 1.44 and 3.79% of the sheep and yaks examined , respectively .
however , the pathogenicity associated with 
 t. multiceps infection in sheep is very high with 97% of the tibetan 
 sheep showing clinical signs dying after developing neurologic disorders due to cerebral 
 coenurosis .
measures should therefore be 
 implemented to prevent infection of dogs both by echinococcus spp . and 
 t. multiceps .
however , 
 previous reports showed that the prevalence of e. multilocularis in humans 
 and dogs was 1.33.3 and 2.53.1% , respectively , in southern qinghai province where chengduo 
 county is located [ 25 , 29 ] .
this parasite is exclusively reported in qinghai - tibetan plateau and maintained by 
 tibetan sand fox and plateau pika .
recently , however , e. shiquicus dna was found in the feces 
 of dogs .
this suggests the possibility of the 
 parasite infecting to dogs , although the coprodna found in dog feces may have been derived 
 from a diet .
mixed infection , i.e. infection with multiple species of taeniid cestodes , was not detected 
 in this study .
however , it should be noted that the method used to identify taeniid species 
 in this study could not always detect cases of mixed infection because the dna examined was 
 obtained from up to 40 eggs . in haiyan county , no eggs were found in the 38 dog feces samples examined .
although an 
 anthelmintic dosing campaign in this county was terminated in 1994 , some of the local 
 residents were aware of the risk of echinococcosis and continued to dose their dogs . 

following a report stating that the prevalence of e. granulosus in yaks and 
 sheep increased after the termination of the campaign , the current prevalence in dogs is unlikely to be 0% ; nonetheless , it is 
 expected to be low .
our observation was contradicted with the previous observation in peru 
 where the termination of a control program may have contributed to a marked increase in 
 infection prevalence in intermediate and definitive hosts as well as in humans [ 17 , 18 ] .
the sensitivity with which parasite eggs , especially cestode eggs , can be detected by fecal 
 examination increases shortly after anthelmintic dosing of animals . in this study ,
taeniid eggs were most frequently detected in dog 
 feces collected 24 hr after dosing .
in the 1 dog from which no taeniid eggs were obtained , a 
 tapeworm ( t. hydatigena ) was excreted after praziquantel dosing . the 
 failure to detect eggs in this animal
may have been due to infection with an immature 
 tapeworm , but we did not check to see if the excreted tapeworm contained any mature eggs 
 ( gravid segments ) . the control trial conducted as part of this study reduced the prevalence of taeniid 
 cestodes in the dogs examined .
however , it was appeared that some of the dog owners did not 
 dose their dogs properly because taeniid cestodes including e. granulosus 
 and t. hydatigena were still detected in 9.6% and 4.9% of the dogs at the 
 end of the 1st and 2nd years of the trial . since the prepatent periods of e. 
 granulosus , t. hydatigena and t. multiceps are 
 about 4560 , 51 and 30 days , respectively , 
 praziquantel doses with 45-day intervals can allow only t. multiceps to 
 become patent infection during the trial .
in addition , the observation that no taeniid eggs 
 were detected in any of the dog feces samples one month after the final examination at the 
 end of 2nd year when all of the dogs were properly dewormed using praziquantel , indicated 
 that praziquantel was highly effective for treating taeniid cestodes . the number of dogs used for the control trial was reduced from 90 to 41 at the end of the 
 trial .
although a variety of reasons were responsible for this decrease in the number of 
 animals , we did not anticipate such a marked reduction at the beginning of the trial .
many 
 of the farmers in the study obtained new dogs to replace those that went missing , and it 
 appears that replacing or trading dogs is common among farmers ; this practice should be 
 therefore considered , if controls are designed in the future .
the questionnaire survey revealed that an awareness of the risk of echinococcosis infection 
 in humans , as well as knowledge of the transmission route of the parasite , would likely 
 affect the efficacy of any control measures .
for example , in heka town , where the control 
 trial of this study was conducted and where local farmers did not know much about 
 echinococcosis , deworming of all of the target dogs was not achieved .
conversely , in haiyan 
 county where a control trial combined with an education program had been conducted 
 previously and where the level of disease awareness among local farmers was high , taeniid 
 eggs were not detected in any of the dog feces samples examined .
these findings suggested 
 that education programs should be an integral component of any measures designed to control 
 canine echinococcosis or initiatives directed at reducing the prevalence of 
 taenia infection in dogs .
 abstractan epidemiological study and control trial were conducted to assess taeniid 
 infection in farm dogs in qinghai province , china . to improve egg detection by fecal 
 examination , a deworming step with praziquantel 
 was incorporated into the sampling 
 methodology . as a result , a marked increase in the number of egg - positive samples was 
 observed in samples collected at 24 hr after deworming . 
 then , the fecal examination and 
 barcoding of egg dna were performed to assess the prevalence of taeniid species in dogs 
 from xinghai , haiyan , gangcha and chengduo counties . 
 analysis of 277 dog feces revealed 
 that taeniid cestodes , including taenia spp . and 
 echinococcus 
 granulosus , were highly prevalent in xinghai ( 34.4% ) , but eggs were not found 
 in haiyan where a control trial on canine echinococcosis had been conducted 20 years 
 previously . 
 a control trial involving the administration of 510 mg / kg praziquantel to 90 
 farm dogs at 45-day intervals was conducted in xinghai . 
 the prevalence of taeniid cestodes 
 in the dogs was reduced to 9.6% and 4.9% after one and two years , respectively , indicating 
 that some dogs were not administered praziquantel properly . 
 a questionnaire survey of 
 farmers in xinghai and haiyan revealed that most farmers in xinghai were not familiar with 
 echinococcosis or the transmission route of the disease , while most farmers in haiyan had 
 a more thorough understanding of the disease . 
 the findings implied that a program for 
 educating local farmers would be important for efficiently controlling canine taeniid 
 infection in the region . 
MATERIALS AND METHODS
RESULTS
DISCUSSION