for the first time in 1911 , it was found as a fungal yeast in human stool specimen ; then , it was identified as a nonpathogenic protozoan and was forgotten for decades ( 2 , 3 ) . between 1970 and 1980 with several studies conducted on blastocystis , the first spark of attraction was paid in relation to biology and clinical features of this parasite ( 4 ) . in recent decades ,
the results of epidemiological studies , in vitro studies , and research on laboratory animals has shown that this parasite is potentially pathogenic ( 3 ) .  
several factors such as parasite load , secreting enzymes such as cysteine protease , parasite subtypes , parasite proteins and even host conditions are involved in the pathogenesis of parasite ( 5 - 8 ) .
blastocystis has a worldwide distribution and is transmitted by cysts via contaminated food and water ( 9 , 10 ) .
its prevalence in developing countries is more than in developed countries , which is a result of poor health ( 11 ) .
isolates of the parasite separated from human are called blastocystis 
 hominis and those from animals are generally called blastocystis sp .
in addition , some classifications may be based on the relevant hosts ( 12 ) . to recognize the genotypes of blastocystis sp .
, a pcr was performed using seven pairs of sequence - tagged sites ( sts ) primers ( 13 ) .
for example , human is the main host of st3 , pig and cattle are the main hosts of st5 , and rodents are the main hosts of st4 ; but these hosts are not specific for blastocystis and the parasite has been reported in human too , which is the sign of zoonotic blastocystis ( 3 ) .
its microscopic diagnosis is difficult so that it is sometimes even ignored by experienced people .
various methods such as direct wet - mount , lugol 's iodine staining , formaldehyde - ether sedimentation , dedicated staining , culture and pcr have been used for its diagnosis ; pcr is the most sensitive method with high specificity ( 14 , 15 ) .
the aim of this study was to determine the subtype of blastocystis in infected cattle in khorramabad city , iran , using seven pairs of sts primers .
it was performed for the first time in iran as an introduction to future studies of subtype prevalence in other animals , also assessing the effect of subtypes on pathogenicity of parasites in animals , particularly in cattle .
this descriptive cross - sectional study was performed on 196 isolates from cattle stool samples collected from slaughterhouse in khorramabad city , iran , in 2012 .
the samples were collected in disposable containers with no fixative . to eliminate waste , 2 g of stool
was added to 10 ml of normal saline solution and the mixture was passed through an 80-micron filter .
afterwards , it was centrifuged for 10 minutes at 200 rpm and 200 mg of the sediment was added to a 1.5 ml microtube ( 16 ) .
dna was extracted using qiaamp dna stool mini kit ( qiagen , hilden , germany ) according to the manufacturer 's protocol .
first , primary primers were used according to previous studies for blastocystis identification ( 14 ) , b11400 forc ( 5`-gga atc ctc tta gag gga cac tat aca t-3 ` ) and b11710 revc ( 5`-tta cta aaa tcc aaa gtg ttc atc gga c-3 ) .
the pcr was performed in a thermocycler ( corbett , australia ) with the following conditions : one initial denaturing cycle at 94c for five minutes , followed by 30 cycles of 94c for one minute , 58c for one minute , and 72c for one minute , and finally one cycle of 72c for five minutes ( 14 ) . at the end
the expected pcr product was 310 bp . to determine the subtype of blastocystis in positive samples , seven pairs of sts primers
the pcr program was conducted with an initial denaturation at 94c for five minutes , followed by 30 cycles of 94c for 40 seconds , 57c for 40 seconds , 72c for 40 seconds , with a final extension at 72c for five minutes ( 13 , 17 ) .
the dna was prepared by pcr , using seven pairs of sts primers and was sent to bioneer corporation , korea , for sequencing .
abbreviations : f , forward ; r , reverse ; st , sequence - tagged .
of 198 specimens , 19 ( 9.6% ) were infected with blastocystis ( figure 1 ) .
three subtypes including st3 , st5 and st6 were found by pcr analysis of the positive samples using the sts primers ( figure 2 ) . among the 19 positive samples , the most common subtype was st5 ( 47.36% ) , followed by st3 ( 10.53% ) and st6 ( 10.53% ) ( figure 2 ) .
st1 , st2 , st4 , and st7 were not identified in the samples under study .
the obtained sequences were compared to the sequences reported in gene bank and the results showed high homology with blastocyctis sp .
nucleotides sequence data with accession numbers cjx524459 , cjx483863 , and cjx524460 respectively for st3 , st5 and st6 have been submitted to the genbank database .
more studies on this parasite have been performed within the recent 10 years and many indices such as morphology , mode of transmission , parasite dissemination , reservoir hosts , and medical importance have been found , while unknown issues like pathogenicity have remained .
studies have been performed on pig and dog in iran to evaluate gastrointestinal parasites such as blastocystis by the microscopy method ( 18 , 19 ) .
this study was the first to investigate the blastocystis subtype in cattle using molecular methods in iran . in the present study , the highest prevalence was for st5 which is so important for epidemiology and risk of human infection .
as has been reported in similar studies , there is a higher risk of st5 existence in animals and humans who live close to them ( 20 - 22 ) .
the main hosts of st5 are pig and cattle ; most of the studies have been conducted on pig for its use in food worldwide ( 23 ) . in iran , due to islamic issues
therefore , people 's contact with cattle is more common than pig . in some studies
, it has been reported that being in contact with animals has provided the transmission of animal subtypes to human ( 24 ) .
the reports of high prevalence of st5 in studies on humans in iran can confirm that this subtype is zoonotic ( 20 - 22 , 25 ) .
however , some researchers believe that st5 is not zoonotic and is found only in pig and cattle ( 23 , 26 ) . on the other hand , st3 reports in cattle as a subtype in human shows the risk of infection transfer between human and cattle . since many of the cattle in this study grazed on farms contaminated with human stool , the zoonotic nature of blastocystis was reconfirmed . another important point in this study
st6 has been reported with mixed infections with st7 in birds . according to this subtype report in this study , further researches and revision of the division of the subtype host are necessary ( 27 ) .
the lack of determination for four subtypes of the 19 positive samples , as reported in previous studies , may be due to genotype diversity , implying that only some of them are known ( 28 ) .
finally , it seems that gathering epidemiological data , ie , identification of zoonotic isolates at the subtype level , is needed for a better understanding of the potential animal reservoirs for human infection .
 background : blastocystis is a zoonotic protozoan parasite living in the digestive system of some vertebrates . 
 this parasite has some subtypes , pathogenicity status of which has still remained controversial.objectives:the aim of this study was to determine the subtype of blastocystis in infected cattle.materials and methods : this descriptive cross - sectional study was performed on 196 isolates from cattle stool samples collected from slaughterhouse in khorramabad city , iran , in 2012 . 
 genomic dna was extracted and to determine the blastocystis subtype , seven pairs of sequence - tagged sites ( sts ) primers were used.results:of 196 specimens , 19 ( 9.6% ) were infected with blastocystis . among the 19 positive samples , the most common subtype was st5 ( 47.36 % ) , followed by st3 ( 10.53% ) and 
 st6 ( 10.53% ) . 
 two ( 10.53% ) samples had mixed infections by st3 and st5 . 
 the four isolates not amplied by any sts primers were probably unknown genotypes.conclusions:in the present study , the highest prevalence was for st5 , which is so important for epidemiology and risk of human infection . the report related to st3 in cattle as a subtype of human showed mutual infection between human and cattle . 
 another important point in this study was the st6 report . 
 finally , it seems that gathering epidemiological data is needed for a better understanding of the potential animal reservoirs for human infection . 