12/06/2012. Background. Taxonomy. Simone M Cacciò. Phylogenetic position of Dientamoeba fragilis

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1 Simone M Cacciò European Union Reference Laboratory for Parasites Department of Infectious, Parasitic and Immunomediated Diseases Istituto Superiore di Sanità 7th Workshop of National Reference Laboratories for Parasites, Rome May, 2012 Background The first description of the parasite was made by Jepps and Dobell in 1918; they initially considered it as a non-pathogenic commensal The name Dientamoeba fragilis refers to the fact that it is an enteric ameba with the curious characteristic of being binucleate and that it tends to degenerate rapidly after excretion in stool Taxonomy Phylogenetic position of Dientamoeba fragilis It has taken a long time to prove that Dientamoeba fragilis is a flagellate that has lost its flagella and not an ameba The parasite is classified within the Parabasalia, and its closest relatives are the Tritrichomonadea (e.g., species of Monocercomonas and Tritrichomonas) 18 S rrna based phylogeny of Parabasalia (Malik et al. PLoS ONE : e20774) 1

2 12/06/2012 They are nice! What are Parabasalia? A monophyletic but complex assemblage of flagellated protists with a characteristic parabasal apparatus (Golgi complex associated with striated fibers) and anaerobic energy-generating organelles called hydrogenosomes. Most parabasalids inhabit the digestive tract of animal hosts as commensals, parasites, or symbionts. Several are of considerable medical and veterinary importance, like Trichomonas vaginalis and Tritrichomonas foetus Structure of Dientamoeba Fine structure of the trophozoite Digestive vacuole Trophozoites showing phagocytosis and engulfing rice starch (Rs) or bacteria (b) Engulfed bacterium Engulfed rice starch Trophozoites dividing by binary fission. Only mononucleated cells divide. Hydrogenosome Pseudopodia Trophozoites appear round after h, when nutrients became less abundant Smooth and ruffled populations of trophozoites observed. Banik et al, IJP 2012, 42: Dientamoeba shows some of the typical structures seen in other parabalids Banik et al, IJP 2012, 42:

3 A rather mysterious organism Life cycle: unknown Life cycle / transmission routes Transmission mode: unknown Natural host range: poorly invesigated Diagnosis: difficult Pathogenicity: controversial Animal model: unavailable Axenic culture: unavailable INDIRECT TRANSMISSION The role of Enterobius as a mechanical vector has been proposed but it is no longer considered as a valid hypothesis DIRECT TRANSMISSION The high frequency of co-infection with other enteric protozoa transmitted through the fecal-oral route suggests that direct transmission could occur. Epidemiology Worldwide distribution of the infection. High prevalence in developing and industrialised countries Between 6 and 30% of people suspected of suffering from intestinal parasitosis will harbor Dientamoeba. Host range Non-human primates (Stark et al., 2008) Pigs (Crotti et al., 2007) From Barrat et al., Gut Microbes 2011, 2:3-12 The limited host range detected suggests human infection may not involve transmission from other animal species. 3

4 Diagnostic methods Microscopy Pleiomorphic trophozoite, 5 to 15 µm in diameter, one to four nuclei, with fragmented chromatin and pale grey-blue finely vacuolated cytoplasm. Diagnostic methods Molecular methods In the last few years, several methods based on PCR have been developed. PCR-RFLP PCR and sequencing Real-time PCR The infection is difficult to diagnose, because of the fragile nature and intermittent shedding of the trophozoites The most commonly employed fixative is SAF (sodium acetate-acetic acid-formalin), whereas staining with iron-haematoxylin or Giemsa is routinely used Both for diagnostics and for identification of genotypes Mostly for diagnostics Two genotypes can be distinguished. Genotype 1 is by far the most commonly found in humans Controversial Pathogenicity We lack an animal model to understand this aspect A rather mysterious organism Life cycle: unknown Transmission mode: unknown Natural host range: unknown Two lines of evidence support the concept that D. fragilis is a true pathgen: Most patients have diarrhea and abdominal pain Treatment of patients resolves clinical symptoms Pathogenicity: controversial Animal model: unavailable Axenic culture: unavailable 4

5 Our study 152 fecal samples from pigs raised in 9 indoor farms of central Italy, 7 in the Umbria region and 2 in the Marche region. Samples from piglets (n = 74, on 8 farms), fattening pigs (n = 14, on 2 farms) and sows (n = 64, on 7 farms). Samples from 21 pig farmers were collected from 5 of the 9 farms, Results of microscopy Farm Herd type Piglet positive / tested 1 Weaner production Fattening pig positive / tested Sow positive / tested Human positive / tested 10 / 10 -/- 1 / 10 -/- 2 Farrow-to-finish 9 / 10 -/- 3 / 10 0 / 4 3 Farrow-to-finish 10 / 10 7 / 10 0 / 10 2 / 8 4 Farrow-to-finish 1 / 10 -/- 0 / 10 -/- 5 Farrow-to-finish 4 / 10 -/- 0 / 10 0 / 2 6 Farrow-to-finish 4 / 10 -/- 1 / 10 -/- 7 Fattening -/- -/- 3 / 4 -/- 8 Fattening 10 / 10 -/- -/- 2 / 3 9 Farrow-to-finish 4 / 4 4 / 4 -/- 0 / 4 Total 52 / / 14 8 / 64 4 / 21 Blastocystis spp. (42%), Endolimax nana (32%) and Iodoamoeba buetschli (25%) were also identified in pigs Molecular typing Only ribosomal sequences are known Diagnostic real-time PCR 18 S rrna ITS S ITS 2 Conventional PCR 28 S Diagnostic real-time PCR Of the 17 human fecal samples, 13 were positive, with cycle threshold (Ct) values ranging from 29 to 40. All 24 microscopically positive pig samples were amplified, with Ct values ranging from 30 to 34, whereas the 14 microscopically negative pig samples were all negative by qpcr. Single and nested PCR for the amplification of various fragments of: 18S rrna ITS S ITS S followed by sequencing of PCR products 5

6 Results at the 5.8S rrna gene The expected size (98 bp) was confirmed by capillary gel electrophoresis Sequence analysis revealed genotype 1 in both pigs (n=11) and humans (n=4) isolates Results at the 18S rrna gene Fixed differences between genotypes 1 and 2 were confirmed by sequencing 18S rrna from 4 human isolates Sequence analysis of nested PCR (366 bp) revealed genotype 1 in all samples, and a very limited amount of polymorphism among isolates from both hosts Sequence comparison by BLAST identified genotype 1 in all samples In 3 pig samples microscopically negative for Dientamoeba, nested PCR and sequencing identified another flagellate, possibly Trichomitus rotunda. Therefore, this PCR assay needs to be optimized in terms of specificity Results at the ITS locus Windsor et al (2006) showed extensive variation between copies of the Internal Transcribed Sequence (ITS) within the same strain. Complex and aberrant chromatograms are observed after direct sequencing of PCR products. Results at the ITS 1 locus To circumvent this problem, it has been proposed to visualise only the Cytosine residues of the ITS1 sequence, and to compare the so-called C-profiles from different strains Overlapping sequences Variable number of T residues due to the presence of several alleles 6

7 Results at the ITS1 locus However, we have been unable to generate reliably specific amplification products of the ITS 1 locus from pig samples, due to interference of DNA from other flagellates present in the feces Nevertheless, the two ITS1 sequences from pigs that could be analyzed were identical to ITS1 sequences obtained from human cases in the United Kingdom and in the Netherlands. Unfortunately, a direct comparison of parasites from pigs and humans from a single Italian farm was not possible. Results at the ITS2 locus On the contrary, specific amplification of the ITS2 locus was obtained from all human and pig samples tested (n=26). Like for ITS1, the ITS2 sequences from PCR products cannot be compared directly. We generated G profiles in the same way as the C profiles, and compared profiles of D. fragilis from humans and pigs P44 P56 P26 c c c Results at the ITS2 locus In this analysis, most isolates appear to have unique G profiles. However, comparative genotyping using ITS1 and ITS2 sequences does not appear, in our opinion, to be a robust method. C or G profiles, indeed, are nut fully reproducible. Moreover, it is unclear on what basis two isolates can be considered genetically identical. There are both qualitatitive (number of alleles) and quantitative (frequency of alleles) aspects Conclusions We have shown that pigs are a natural host of Dientamoeba fragilis We confirmed that piglets are more susceptible to infection Molecular analyses of 5.8S and 18S rrna sequences demonstrated that pigs harbor genotype 1, as humans Comparative analyses of ITS1 and ITS2 sequences are difficult 7

8 Perspectives The possibility of zoonotic transmission raised by our results deserves future investigations THANK YOU FOR YOUR ATTENTION! There is a need to develop molecular markers suitable for comparative studies The availability of an animal model will be very useful to study various aspects of the biology of this elusive organism QUESTIONS? 7th Workshop of National Reference Laboratories for Parasites, Rome May,