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 .
 the world is home to more than 1 billion pigs , which produce large quantities of feces . we know that some organisms in pig feces can cause human disease , and now we might have another to add to the list . 
 little is known about where the common intestinal parasite dientamoeba fragilis comes from and how it is spread . 
 however , recent molecular analysis confirmed that the organism found in pigs is indeed the same as the one found in humans . 
 therefore , pigs ( or their feces ) might be a source of this parasitic infection in humans . 
The Study
Conclusions