Pigs as Natural Hosts of Dientamoeba fragilis Genotypes Found in Humans

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.

Dientamoeba fragilis is a common intestinal parasite in humans. Transmission routes and natural host range are unknown. To determine whether pigs are hosts, we analyzed 152 fecal samples by microscopy and molecular methods. We confi rmed that pigs are a natural host and harbor genotypes found in humans, suggesting zoonotic potential.
T he fl agellated protozoan Dientamoeba fragilis is one of the most common parasites in the intestinal tract of humans (1). Infection is highly prevalent in economically developing regions and in industrialized countries (1,2). Infected persons often show no symptoms, but a pathogenic role for this parasite has been reported recently in humans and gorillas (2)(3)(4). Little is known about transmission routes of this parasite, and a transmissible stage (e.g., a cyst) has not been described (1,5). Molecular characterization of human isolates based on sequence analysis of ribosomal genes revealed 2 genotypes (1 and 2), with genotype 1 predominating worldwide (6,7).
Other than humans, few animal hosts of D. fragilis have been reported. Surveys of mammals and birds have identifi ed only nonhuman primates (gorillas, macaques, and baboons) as natural hosts (8,9). Recently, however, a high prevalence of infection (43.8%) has been reported in pigs in Italy (10). To determine whether pigs are a host of D. fragilis, we analyzed fecal samples from 152 pigs in Italy by microscopy and molecular methods.

The Study
During June-August 2010, a total of 152 fecal samples were collected from the rectums of piglets (age 1-3 months; weight 6-24 kg), fattening pigs (age 3-4 months; weight 25-50 kg), and sows (age 1-2 years; weight 180-250 kg). The pigs were raised in 6 farrow-to-fi nish 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). Pig fecal samples from 7 of the 9 farms were available for molecular analysis. 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 μm to 20 μm, with fragmented chromatin and pale gray-blue fi nely 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.
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). The microscopic analysis also showed Blastocystis spp. (in 42% of pigs), Endolimax nana protozoa (32%), Iodoamoeba buetschli protozoa (25%), and other fl agellates (4.5%). Of the 21 samples from pig farmers, 4 from farmers working on 2 farms were positive for D. fragilis by microscopy (  Table 2). Genotype 2 was not found in any of the samples from pigs or humans. 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 confi rmed by sequencing the entire 18S rRNA gene from 2 reference isolates and 2 human isolates from this study. Amplifi cation was obtained from 6 of the 24 positive pig samples and from 8 of the 17 human samples. Genotype 1 was identifi ed in all samples (Figure 2). 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 fl agellate of reptiles, although the sequence could originate from T. rotunda, a fl agellate of pigs that has not been described at the molecular level.
Last, we studied the more variable ITS1 locus. Amplifi cation was obtained from 11 of the 24 pig samples ( Table 2), but only 2 sequences could be clearly identifi ed as D. fragilis. Four sequences showed homology (80%) with fl agellates from different vertebrate classes whereas the remaining 5 sequences were excluded because of insuffi cient 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 amplifi ed 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).

Conclusions
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 diffi culties 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   Pig 6  1  --ND  -ND  -ND  Pig 7  1  --ND  -ND  -ND  Pig 8  1  --ND  -ND  -ND  Pig 9  1  --ND  -ND  -ND  Pig 10  1  --ND  -ND  -ND  DF-P1  6  --ND  -ND  -ND  DF-P2  6  --ND  -ND  -ND  DF-P3  6  --ND  -ND  -ND  DF-P4  6  --ND  -ND  -ND  H1 2 †  -ND  H12  3  -+  ND  -ND  -ND  H13  3  -+  ND  -ND  -ND  H14  3  --ND  -ND  -ND   The opinions expressed by authors contributing to this journal do not necessarily refl ect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affi liated.