study sites : the study sites were yushu county at yushu state and maqin 
 county at guoluo state located in southeast qinghai ; guinan county at hainan state in 
 middle - east qinghai ; and haiyan county at haibei state in qinghai lake area ( fig . 1fig .
investigation of the prevalence of hypodermosis in yaks : the investigation 
 was carried out in yushu , maqin and haiyan counties between march and may in 2008 .
the skin 
 surfaces of the neck , shoulder , chest and back of 25,769 live yaks were examined for 
 hypoderma lumps by hand palpation .
the lumps were counted , and the 
 prevalence of hypoderma larval infection and the mean number of lumps in 
 different age groups ( 1- , 2- and 3-year - old and adult ( 4-year - old ) ) of yaks were evaluated 
 for each county .
investigation of seasonal pattern of hypoderma larval development and migration in 
 yak bodies : the investigation was performed in two counties .
one was yushu county 
 with an average elevation of 4,000 m , and the other was haiyan county with an average 
 elevation of 3,000 m. every month from april 2008 to march 2009 , five one - year - old yaks were 
 selected from the herd grazed in the counties and autopsied to check for 
 hypoderma larvae in visceral organs , such as esophagus , rumen , jejunum , 
 colon , diaphragm , spinal canal and in the skin of back .
the larvae collected were fixed in 
 10% formalin and counted , and their instar larval stage was determined as previously 
 described [ 10 , 15 ] .
determination of an effective dose of ivermectin for hypoderma larvae : in 
 yushu , maqin and haiyan counties , different doses of ivermectin ( beijing zhongnong huawei 
 pharmaceutical co. , ltd . , beijing , china ) were administered to yaks younger than 2-year - old 
 in october 2008 .
a liquid ivermectin formula was put into a syringe and splashed onto the 
 yaks back .
the yaks were divided into four groups ( 1,0001,500 head / group in each county ) ; 
 the full dose group was given a single treatment with 500 g / kg body weight 
 ( bw ) which is the dose recommended for the treatment of hypoderma larvae by 
 the commercial suppliers ; the half dose group was given 250 g / kg bw , the 
 quarter dose group was given 125 g / kg bw and the control group remained 
 untreated . between february and april 2009 , the yak s skin surface was hand - palpated to 
 check and count hypoderma lumps .
the neck , shoulder , chest and back were 
 examined . conducting a large - scale control trial : between 2009 and 2011 , a 
 large - scale control trial against hypoderma
larval infection in yaks was 
 carried out in four counties ( maqin , yushu , guinan and haiyan ) .
in each county , four age groups ( 1- , 2- and 3-year - old and adult ( 4-year - old ) ) 
 of yaks were treated with 125 g /kg bw of ivermectin by splashing onto the 
 back of yaks in october .
then , in march and may of the next year , approximately 10% of the 
 treated yaks were selected randomly , and their skin surfaces were hand - palpated to check and 
 count hypoderma lumps . the neck , shoulder , chest and back were examined . 

the proportion of yaks used for this study was about 2 to 5% of the total population of the 
 grazed yak herd in each study site .
statistics : ninety - five percent confidence intervals ( 95% ci ) of 
 prevalence were calculated on the basis of binomial distributions using the software program 
 r .
prevalence of hypodermosis in yaks : the prevalence in different age groups 
 of yaks in yushu , maqin and haiyan counties is shown in table 1table 1.prevalence of hypoderma larval infection in yaks of different 
 ages in three counties of qinghaicountyage groupno .
infectedprevalence ( % ) ( 95% ci)mean no . lumpsyushu1-year - old1,9781,90296.2 ( 95.296.9)13.52-year - old2,2462,12494.6 ( 93.695.4)11.23-year - old2,5802,15683.6 ( 82.184.9)10.9adult3,2641,43043.8 ( 42.145.5)9.8total10,0687,61275.6 ( 74.876.4)11.4maqin1-year - old2,0071,91195.2 ( 94.296.1)12.42-year - old2,1512,02394.0 ( 93.395.0)10.23-year - old2,0801,71082.2 ( 80.583.8)11.4adult2,6671,13042.4 ( 40.544.3)8.2total8,9056,77476.1 ( 75.276.9)10.8haiyan1-year - old2,3402,25096.2 ( 95.396.9)14.42-year - old1,8801,85598.7 ( 98.099.1)15.53-year - old1,5531,38689.2 ( 87.690.7)15.2adult1,02351850.6 ( 47.653.7)9.8total6,7966,00988.4 ( 87.689.2)14.5a ) 95% confidence interval .
b)mean number of hypoderma lumps 
 found per yak .. all counties showed a similar pattern of age difference in prevalence . in all 
 counties ,
the prevalence in 1- and 2-year - old yaks was very high ( more than 94% ) . in 
 comparison ,
the number of 
 hypoderma lumps detected per yak was also higher in young ( 1- to 
 3-year - old ) yaks than that in adult yaks .
it is recognized that the prevalence and the mean 
 number of lumps were relatively higher in haiyan county than other counties .
seasonal pattern of hypoderma larval development and migration in the yak 
 body : the number of larvae and their instar larval stages in the yaks at yushu 
 and haiyan counties in each month from april 2008 to mach 2009 are shown in table 2table 2.monthly change in the mean number of 1st , 2nd and 3rd instar 
 hypoderma larvae found in yaks in two counties of qinghai between 
 april 2008 and march 2009yearmonthyushu countyhaiyan county1st instar2nd instar3rd instar1st instar2nd instar3rd instar2008apr 0045.80046.6may 0030.20032.8june002.2003.4july000000aug26.20025.000sep40.20038.800oct116.000102.400nov57.824.2058.837.60dec17.663.2017.265.402009jan12.27517.810.269.016.6feb2.837.846.23.639.851.6mar0066.60065.0 . a quite similar pattern was observed in both counties .
first instar larvae 
 appeared in the yak bodies in mid - august 2008 and were continually found until february 
 2009 .
the number of 1st instar larvae detected per yak was highest in october 2008 and then 
 decreased gradually .
the migration pattern of 1st instar larvae in yak bodies was quite 
 similar in the two counties , and thus , the data are combined and shown in table 3table 3.the mean number of 1st instar hypoderma larvae detected in 
 various organs of yaks in yushu and haiyan counties of qinghai between august 2008 and 
 february 2009yearmonthesophagusrumensmall intestinecolondiaphragmspinal canal2008aug8.87.64.63.20.50.9sep17.116.35.00.80.3oct56.253.0nov31.526.82009jan9.97.5feb7.33.9 .
the majority of larvae were found in the subserosa of the esophagus and 
 rumen .
however , although restricted in august and september , parts of the larvae were also 
 found in the subserosa of the small intestine and colon and in the diaphragm and spinal 
 canal . in november 2008 ,
the number of 2nd instar larvae detected per yak increased in december 2008 , peaked in 
 january 2009 and disappeared by march .
all of the larvae were found in the subcutaneous 
 tissue of the back ( table 2 ) . in january 2009
, 3rd instar larvae appeared on the back of animals , and the number detected 
 per yak peaked in march 2009 . since 3rd instar larvae were found from april to june 2008 , it 
 is suspected that they parasitize yaks between january and june ( table 2 ) .
third instar larvae could easily be distinguished from 2nd instar larvae by the body size 
 and color .
the 2nd instar larvae were 1115 mm long and 36 mm wide , while 3rd instar larvae 
 were 2330 mm long and 1215 mm wide .
the body color of 2nd instar larvae was yellowish 
 white , while that of 3rd instar larvae was yellowish to dark brown .
an effective dose of ivermectin for hypoderma larvae : the effect of 
 different doses of ivermectin administration on hypoderma larval infection 
 in yaks is shown in table 4table 4.the effect of administration of different doses of ivermectin on 
 hypoderma larval infection in yakscountydoseno .
lumps*yushu500 g / kg1,100000250 g / kg1,058000125 g / kg1,35020.21.5control1,06499293.212.9maqin500 g / kg1,500000250 g / kg1,258000125 g / kg1,05010.11.0control1,1641,09293.212.6haiyan500 g / kg1,800000250 g / kg1,558000125 g / kg1,25020.21.0control1,3641,29094.613.7*mean number of hypoderma lumps found per yak .. the trials conducted in all three counties ( yushu , maqin and haiyan ) showed 
 similar results .
the prevalence in the control groups was very high ( 93.294.6% ) , and the 
 mean number of lumps found was around 13 .
in contrast , no hypoderma lumps 
 were found in the groups treated with 500 and 250 g / kg bw ivermectin , and 
 hence , no infected animals were found .
the prevalence in the groups treated with 125 
 g / kg bw ivermectin was also extremely low ( 0.10.2% ) , and the mean 
 number of lumps found was 1.0 to 1.5 .
* mean number of hypoderma lumps found per yak . a successful control of wable fly larval infection : cumulative data from 
 the trial conducted over three years in four counties are shown in table 5table 5.prevalence of hypoderma larval infection and mean number of 
 lumps found in different age groups of yaks after administration of ivermectin in four 
 counties in qinghaicountyage groupno . treatedno .
lumps*yushu1-year - old38,0503,797340.91.22-year - old37,5423,916350.91.13-year - old32,0393,114270.91.0adult25,8732,561220.91.0maqin1-year - old44,6444,576451.01.12-year - old40,0214,083350.91.43-year - old31,9433,306240.71.1adult24,4342,592140.51.1haiyan1-year - old42,3164,483370.81.12-year - old40,0344,206350.81.03-year - old30,6183,158190.61.0adult23,4632,486130.51.0guinan1-year - old51,7225,282430.81.22-year - old43,7204,535370.81.23-year - old31,9343,266240.71.4adult24,6422,612180.71.0*mean number of hypoderma lumps found per yak .. there was no obvious difference in the annual data between 2009 and 2011 in 
 the four counties .
some yaks remained infected in all counties and in all age groups , but 
 the overall prevalence was < 1.0% and the mean number of lumps found was below 1.5 at 5 to 
 7 months post - administration of ivermectin . there was no obvious difference among the data 
 obtained from different age groups or different counties . *
this survey was conducted in four counties located in the middle - east qinghai to qinghai 
 lake area ( haiyan county and guinan county ) and southeast qinghai area ( yushu county and 
 maqin county ) .
these two areas are 1,000 km apart with average elevation of 3,000 and 4,000 
 m , respectively .
they have different ecological environments with annual fluctuations in 
 mean atmospheric pressure , temperature and precipitation that could affect the distribution 
 of hypoderma .
however , we did not find any obvious differences in the 
 prevalence of hypoderma in yaks from these two areas .
the results indicate 
 that hypoderma is distributed widely in the east qinghai - tibetan 
 plateau .
it should be emphasized that the prevalence in young yaks was high ( > 94% ) compared to 
 adult yaks which was around 50% .
the reason for this observation is not clear , but it could 
 be partially explained by difference in the movement constraint used in young and adult 
 yaks .
thus , young 
 yaks are tethered to stakes so that they can not move and drink milk freely from their 
 mothers .
in contrast , adult yaks were able to move freely and escape from adult warble fly 
 attack by swishing their tails or running away .
therefore , it would be relatively difficult 
 for warble flies to lay their eggs on adult yaks .
also , acquired resistance against 
 hypoderma infection may play a significant role in the infection in adult 
 yaks [ 10 , 16 ] . the seasonal development and migration patterns of hypoderma larvae in yak 
 bodies in the study areas
it has been reported that the 1st instar 
 larvae of h. lineatum and h. sinense parasitized in the 
 esophagus and those of h. bovis in the epidural fat of the spinal canal 
 [ 2 , 4 , 15 , 21 ] .
however , 
 as observed in our previous study , a large 
 proportion of 1st instar larvae were found not only in the esophagus but also in the rumen . 

moreover , the larvae were detected in other organs in late summer to early autumn ( august 
 and september ) .
although morphological identification for the species of 1st instar larvae is impossible 
 , dna identification of species would help to 
 understand the migration patterns of h. bovis , h. lineatum and h. 
 sinense in yaks .
hypoderma in the study areas showed the similar seasonal larval 
 development , and therefore , ivermectin was administered at the same time ( october ) to yaks 
 grazing the different sites .
the timing of the ivermectin administration was chosen based on 
 our previous research that showed that administration in october resulted in complete 
 elimination of the larvae from yaks , but that administration in september or november 
 resulted in incomplete elimination .
ivermectin has been previously shown to be effective for deworming 
 hypoderma larvae [ 1 , 7 , 11 , 14 , 18 , 20 ] . however , the continuous use or overuse of ivermectin 
 could be accompanied by side effects to the animals treated [ 5 , 9 ] , environmental pollution , induction of drug - resistant flies or impairment of 
 human health as a result of consumption of meat containing the chemical .
therefore , it is ideal to use the minimum effective 
 dose of ivermectin with the minimum number of treatments per year .
this study showed that a single administration of full and half 
 doses of ivermectin could completely eliminate the parasites from yaks .
a single quarter 
 dose administration was also quite effective and eliminated most of the larvae from yaks . 

therefore , we determined that a single dose of 125 g / kg bw of ivermectin 
 was an optimal dose for treatment .
as expected , a large - scale control trial conducted in this study was successful .
although 
 complete elimination of hypoderma larvae from treated yaks was not achieved 
 and a small number of hypoderma lumps remained in some yaks after 
 treatment , the trial showed a high cost - performance ratio .
we believe that the following 
 factors may also have contributed to the successful control . before implementation of this 
 project , a training program for the tibetan local nomads
was conducted to teach the method 
 and the appropriate time of year for administration of ivermectin to yaks .
in addition , 
 since restraining yaks is very difficult , administration of the drug by splashing onto the 
 animals seemed to be a suitable method which is also less laborious , easier and safer than 
 injection . in conclusion ,
the control method used in this study would be suitable for use in 
 the qinghai - tibetan plateau to reduce economic losses due to hypoderma 
 infection and contribute to the improvement of animal health and subsequently human 
 health .
we assumed multiple species of hypoderma were detected in this study , 
 because we had detected two species of hypoderma , h. bovis 
 and h. lineatum , in yaks at the same sites of this study in a previous 
 study ( unpublished ) .
however , regrettably , we did not identify the species of 
 hypoderma detected in the present study . because the difference in the 
 species construction could affect the local epidemiology and larval migration pattern , 
 species identification by morphological and dna sequence characteristics
 abstractthe prevalence of hypoderma spp . in yaks grazed in the east of qinghai 
 province was investigated in 2008 . in this area , 
 the prevalence in young yaks ( 1- to 
 3-year - old ) was very high at 82.298.7% , whilst in adult yaks ( 4-year - old and older ) , the 
 prevalence was 42.450.6% . 
 the seasonal development and migration pattern of 
 hypoderma larvae in yak bodies was found to be similar for different 
 locations in this area . 
 the numbers of first , second and third instar larvae detected in 
 yak bodies peaked in october , december and march , respectively . 
 different doses of 
 ivermectin ( 125 to 500 g / kg body weight ) almost completely dewormed the 
 larvae from yaks , suggesting that using a quarter of the prescribed dose ( 500 
 g / kg body weight ) was effective . 
 in october of each year between 2009 
 and 2012 , ivermectin ( 125 g / kg body weight ) was administered to a total 
 of 562,995 yaks grazed in four counties in qinghai province , and the pevalence of 
 hypoderma larval infection in yaks was reduced to 0.51.0% . 
MATERIALS AND METHODS
RESULTS
DISCUSSION