during june  august 2010 , a total of 152 fecal samples were collected from the rectums of piglets ( age 13 months ; weight 624 kg ) , fattening pigs ( age 34 months ; weight 2550 kg ) , and sows ( age 12 years ; weight 180250 kg ) . the pigs were raised in 6 farrow - to - finish farms , 2 fattening farms , and 1 weaner indoor farm of central italy ( 7 farms in the umbria region and 2 farms in the marche region ) . fecal samples from 21 pig farmers were collected from 5 of the 9 farms , 17 of which were available for molecular analysis . microscopic diagnosis of d. fragilis was based on visualization of pleomorphic trophozoites , ranging in size from 4 m to 20 m , with fragmented chromatin and pale gray - blue finely vacuolated cytoplasm after giemsa staining ( figure 1 ) . dna was extracted directly from 200 mg of feces by using the qiaamp dna stool minikit ( qiagen , hilden , germany ) . reference d. fragilis dna of genotype 1 ( strains 379 and 1085 ) was used as a positive control . dientamoeba fragilis trophozoites in a smear of pig feces after giemsa staining , italy , 20102011 . a taqman real - time pcr that targets the 5.8 s ribosomal locus was performed in a lightcycler 480 apparatus ( roche diagnostics gmbh , mannheim , germany ) as described ( 11 ) . for the 18s rrna gene , a published assay ( 12 ) was used to amplify a 662-bp fragment , followed by amplification of a 366-bp fragment with newly designed primers df322for ( 5-gagaaggcgcctgagagata-3 ) and df687rev ( 5-ttcatactgcgctaaatcatt-3 ) . for the internal transcribed spacer 1 ( its1 ) region , the forward primer ssu2 ( 13 ) and the reverse primer df - itsrev ( 5-gcgggtcttcctatataaacaagaacc-3 ) were used , whereas the forward primer df - itsnesfor ( 5-atacgtccctgccctttgta-3 ) and the reverse primer df - itsnesrev ( 5-gcaatgtgcattcaaagatcgaac-3 ) were used in the nested pcr . the sequences were assembled by using seqman ii ( dnastar , madison , wi , usa ) and compared with those available in public databases by using blast ( http://blast.ncbi.nlm.nih.gov/blast.cgi ) . the microscopic examination showed that 52 of the 74 piglets , 11 of the 14 fattening pigs , and 8 of the 64 sows were positive for d. fragilis ( table 1 ) . more trophozoites were observed in fecal samples from piglets , suggesting a higher susceptibility of young animals to infection ( data not shown ) . ( in 42% of pigs ) , endolimax nana protozoa ( 32% ) , iodoamoeba buetschli protozoa ( 25% ) , and other flagellates ( 4.5% ) . of the 21 samples from pig farmers , 4 from farmers working on 2 farms were positive for d. fragilis by microscopy ( table 1 ) . molecular techniques were applied to 38 pig fecal samples , specifically 24 samples positive by microscopy from 6 farms and 14 samples negative by microscopy from 2 farms , and to all 17 human fecal samples . a comparison of human and pig samples collected from the same farm was possible for farms 2 , 3 , and 5 ( table 2 ) . using real - time pcr , all 24 positive pig samples were amplified , with cycle threshold values ranging from 30 to 34 , whereas none of the 14 negative samples were positive to this assay ( table 2 ) . however , because no inhibition controls were run , false - negative results can not be ruled out . of the 17 human fecal samples , 13 were positive with cycle threshold values of 2940 . the sequence analysis of 15 amplified products ( 11 from pigs and 4 from humans ) showed 100% homology with d. fragilis genotype 1 ( table 2 ) . genotype 2 was not found in any of the samples from pigs or humans . * # 100% identity to dq223442 , dq223450 , dq223452 , dq223454 , dq223456 , and dq167586 . next , a 366-bp fragment of the 18s rrna gene was analyzed . in this fragment , genotypes 1 and 2 can be distinguished by 8 substitutions or insertions or deletions ( figure 2 ) , which were further confirmed by sequencing the entire 18s rrna gene from 2 reference isolates and 2 human isolates from this study . amplification was obtained from 6 of the 24 positive pig samples and from 8 of the 17 human samples . one human isolate ( h7 ) showed a single nucleotide substitution in the fragment sequenced ( figure 2 ) . sequences from 3 microscopically negative pig samples ( all from farm 1 ) had a high homology ( 96% ) with trichomitus batrachorum , a flagellate of reptiles , although the sequence could originate from t. rotunda , a flagellate of pigs that has not been described at the molecular level . multiple alignment of the 366-bp fragment of the 18s rrna gene from dientamoeba fragilis genotypes 1 and 2 . amplification was obtained from 11 of the 24 pig samples ( table 2 ) , but only 2 sequences could be clearly identified as d. fragilis . four sequences showed homology ( 80% ) with flagellates from different vertebrate classes whereas the remaining 5 sequences were excluded because of insufficient quality . the 2 d. fragilis sequences from pigs showed 100% homology with sequences from human isolates from the united kingdom ( table 2 ) , further supporting the presence of genotype 1 in these 2 hosts . a direct comparison of its1 sequences from humans and pigs from a single farm in italy was not possible because d. fragilis was amplified from only 2 human samples from 2 farms from which no pig samples were available . the analysis of its1 from the 2 human isolates showed full identity to human isolates from the netherlands and the united kingdom ( table 2 ) . considering the size of the world s pig population ( > 1 billion ) , the close contact between pigs and humans in many parts of the world , and the difficulties in the proper management of pig fecal waste , the role of these animals as reservoirs of zoonotic pathogens must be carefully evaluated . we demonstrated that pigs are hosts of d. fragilis , on the basis of molecular analysis of 3 fragments in the ribosomal cluster . sequence analyses of fragments of the 18s and 5.8s rrna genes showed genotype 1 in isolates collected in the same farm from humans and pigs , suggesting the potential for zoonotic transmission of this parasite . if a transmissible cyst stage exists , then environmental contamination with pig feces should be considered a key factor in the transmission of this parasite . pigs also are a fascinating animal model to elucidate the life cycle of this elusive parasite .