Establishment of a continuous culture system forEntamoeba murisand analysis of the small subunit rRNA gene

Abstract

Summary :We established a culture system for Entamoeba muris (MG-EM-01 strain isolated from a Mongolian gerbil) using a modified Balamuth's egg yolk infusion medium supplemented with 4 % adult bovine serum and Bacteroides fragilis cocultured with Escherichia coli. Further, encystation was observed in the culture medium. The morphological characteristics of E. muris are similar to those of Entamoeba coli (E. coli); moreover, the malic isoenzyme electrophoretic band, which shows species-specific electrophoretic mobility, of E. muris had almost the same mobility as that observed with the malic isoenzyme electrophorectic band of E. coli (UZG-EC-01 strain isolated from a gorilla). We determined the small subunit rRNA (SSU-rRNA) gene sequence of the MG-EM-01 strain, and this sequence was observed to show 82.7 % homology with that of the UZG-EC-01 strain. Further, the resultant phylogenetic tree for molecular taxonomy based on the SSU-rRNA genes of the 21 strains of the intestinal parasitic amoeba species indicated that the MG-EM-01 strain was most closely related to E. coli. E ntamoeba muris is a highly contagious intestinal protozoan parasite of laboratory mice, rats and other rodents; this species is morphologically similar to Entamoeba coli (E. coli) (Neal, 1950) and primarily proliferates and encysts in the caecum of mice (Lin, 1971). In vitro culture of E. muris has been attempted (Neal, 1950;Simitch & Petrovitch, 1951;Smith et al., 1985); however, trials to achieve successive culture have not been successful. In the present study, we established a system for the stable and successive culture of an E. muris strain. Further, we attempted phylogenetic analysis of this strain to help in investigating its molecular taxonomy. Résumé MATERIALS AND METHODS E.muris (strain MG-EM-01) isolated from a Mongolian gerbil spontaneously infected in our laboratory was used to establish the culture system. An E. coli strain (UZG-EC-01) isolated from a gorilla in a zoo in Tokyo, Japan, was used as a reference. Escherichia coli and Bacteroides fragilis strains were isolated from the stool of a primate [DeBrazza's guenon (Cercopithecus neglectus)] and fresh human stool samples from a patient with intestinal amoebic colitis, respectively. The E. coli strains were maintained in a chemically defined medium (R medium) (Robinson, 1968), and the B. fragilis strains were maintained on trypticase, yeast extract and iron (TYI) broth (Diamond et al., 1978); these strains were used as supplements for the culture system of E. muris.Article available at http://www.parasite-journal.org or http://dx.doi.org/10.1051/parasite/2009162135The egg yolk infusion medium of Balamuth's medium (Balamuth, 1946) was replaced with infusion of a liver concentrate with 4 % preheat-treated adult bovine serum (56°C for three hours). At least one day prior to E. muris culture, 0.1 ml of E. coli maintained in the R suspension was added from the stock culture preserved at 4°C (used within one month); however, 0.2 ml of a 1-to 3-day culture of B. fragilis maintained in ...

Expanding the Entamoeba Universe: New Hosts Yield Novel Ribosomal Lineages

“…Furthermore, there is only one reliable GenBank entry for E. muris , isolated from Mongolian gerbil (Kobayashi et al. ); the other sequence listed as E. muris (), isolated from Rattus rattus , shares 100% identity with E. coli ST2, also an 8‐nucleated cyst former, and is likely to have been misidentified because of the host. There are no GenBank sequence entries for either E. funamulae or E. citelli .…”

“…Only recently has PCR been used to detect and differentiate E. histolytica and E. dispar in rats, which were shown by phylogenetic analysis of partial SSU rDNA sequences to cluster with those typically found in primates (Lau et al 2014). Furthermore, there is only one reliable GenBank entry for E. muris, isolated from Mongolian gerbil (Kobayashi et al 2009); the other sequence listed as E. muris (FN396613), isolated from Rattus rattus, shares 100% identity with E. coli ST2, also an 8-nucleated cyst former, and is likely to have been misidentified because of the host. There are no GenBank sequence entries for either E. funamulae or E. citelli.…”

Further insight into the genetic diversity of Entamoeba coli and Entamoeba hartmanni

“…Based on morphology, E. coli has also been reported from, among others, pigs (Pinilla et al, 2021) and dogs (Campos Filho et al, 2008). In addition to E. coli , octonucleate cyst‐producing species of Entamoeba for which DNA sequences are available include Entamoeba muris (Kobayashi et al, 2009) and Entamoeba sp. RL7 (Jacob et al, 2016).…”

Epidemiology, species composition and genetic diversity of tetra- and octonucleated Entamoeba spp. in different Brazilian biomes

“…Only one haplotype has been previously described, isolated from humans in England (AF149914) [41]. It is hypothesized that the ST1 subtype is more common than ST2 in humans [42], and ST2 was recently identified in wild nonhuman primates in Asia and Africa [43,44]. It is intriguing to observe a large number of new E. coli ST2 subtypes in humans living in the Amazon rainforest, the habitat of great diversity of neotropical primates.…”