MATERIALS AND METHODS

The fresh fecal sample used in this study was collected from a King Horseshoe bat from the Qian-Ling Park, GuiYang, China (10 July 2010). The parasite was cultured in Trypsin-yeast- iron (TYI-S-33) medium axenically at 37°C. After 48 h growth, the culture tubes were placed in ice-cold bath for 5 min and trophozoites were centrifuged, resuspended in phosphate-buffered saline pH 7.2. Trophozoites were harvested and used in the following experiments. The presence of parasites in the fecal sample and culture fluid was determined by microscopic examination (Tanyuksel and Petri, 2003). A commercially available kit (ELISA kit E. histolytica-II; Techlab, Inc., Blacksburg, VA.) was used in this study. This kit is based on the monoclonal antibody-peroxidase conjugate specific for E. histolytica adhesin. According to the manufacturer’s instructions, a positive result was defined as an optical density reading >0.05 after subtraction of the negative control optical density. Trophozoites were centrifuged at 5,000x g for 5 min and washed twice in phosphate-buffered saline. After sodium dodecyl sulfate-proteinase K treatment (2 h at 55°C), the sample was processed further by the conventional phenol-chloroform DNA extraction method (Blin and Stafford, 1976). The PCR was conducted in a volume of 25 μL containing (final concentrations) 20 pmol of each primer specific for E. histolytica and E. dispar (p11 plus p12 and p13 plus p14, respectively) as previously described by Tachibana et al. (1991), Pinheiro et al. (2004) and Zongo et al. (2008). The PCR product was isolated by electrophoresis on 2% agarose gels containing ethidium bromide and the gels were photographed under ultraviolet light. The product was cloned into a pMD18-T plasmid vector (TaKaRa, Japan) according to the manufacturer’s recommendations. The cloned insert was sent to Shanghai Sangon Biological Engineering Technology and Services for sequencing. The obtained DNA sequence was submitted for BLAST searching in Gen-Bank to ensure that the required sequence had been amplified.

Mere use of primers p11 plus p12 in PCR and sequencing is not enough to conclude that the isolate was indeed E. histolytica (Inyang-Etoh et al., 2007; Takano et al., 2007). In this report, we use a different molecular marker to identify whether the isolate was indeed E. histolytica or not. The real-time PCR assay for the detection of E. histolytica was carried out according to a previously described (Roy et al., 2005; Rasti et al., 2006). The performance of the assay was evaluated using a range of control samples including E. histolytica (No. GY10), E. dispar (No. TR327), E. moshkovskii (No. DY12) and E. nuttalli (No. ZY27) (These control samples were isolated and maintained by our research center). Amplification results were analyzed using i-Cycler software, version 3.0 for Windows. Amplification was also confirmed in all reactions by gel electrophoresis. A sample was considered positive if the signal Cycle Threshold (CT) value exceeded a preset threshold. The PCR product was isolated by electrophoresis on 2% agarose gels containing ethidium bromide and the gels were photographed under ultraviolet light. The product was cloned into a pMD18-T plasmid vector (TaKaRa, Japan) according to the manufacturer’s recommendations. The cloned insert was sent to Shanghai Sangon Biological Engineering Technology and Services for sequencing. The obtained DNA sequence was submitted for BLAST searching in Gen-Bank to ensure that the required sequence had been amplified.

Metronidazole was the drug of choice for enteric E. histolytica infections at a dose 5 mg, two times daily orally for 5 days. Fecal samples were examined three times after the treatment on days 5, 10 and 30.

RESULTS

Abundant amoebic cysts were detected in the fresh fecal sample. They were spherical, about 12.0 μm in diameter. Some precysts as the pecilo oblate type were bigger than the metacysts. Lots of amoeba trophozoites and cysts were found in the smear of the culture fluid. The trophozoites had a thick clear layer of ectoplasm and granular endoplasm. They moved rapidly in a straight line with a single clear pseudopod at the anterior end. The endoplasm of the trophozoites contained a profuse fine particle and a vesicular nucleus. The spherical cysts averaged 12.21 μm (n = 100) with a range of 8-16 μm.

Fig. 1:	Agarose gel electrophoresis of PCR products for using primers p11/p12 and p13/p14. Lane A, negative control; Lane B, amplifications for primers p13/14; Lane C, DNA molecular mass standards (DNA Marker |); Lane D, amplifications for primers p11/12

Fig. 2:	The Results of real-time PCR specificity detection. 1: DNA from fresh fecal sample of bat after cultrued; 2: DNA from control sample of E. histolytica, No.GY10; 3: Control group; 4: DNA from control sample of E. dispar (No. TR327); 5: DNA from control sample of E. nuttalli, No. ZY27; 6: DNA from control sample of E. moshkovskii, No. DY12

The cysts contained one to four nuclei as observed with the iodine stain. The E. histolytica/E. dispar microscopically positive sample was identified as E. histolytica using PCR-specific primers P11/P12, demonstrated by amplification of the species-specific fragment (100 bp) (Fig. 1). Conversely, no amplification was observed for nonpathogenic E. dispar using PCR-specific primers P13/P14. Sequencing the PCR amplification fragment of the species specific primers P11/P12 produced a 100-nucleotide base sequence. This determined sequence was the part sequence of E. histolytica peroxiredoxin gene after BLAST searching in Gen-Bank. Simultaneously, the sample was positive in the immunoenzymatic assay for the presence of E. histolytica-specific galactose adhesion.

The amplification plot of the E. histolytica-specific real-time-PCR assay is shown in Fig. 2. The real-time-PCR assay was specific for E. histolytica detection, as it was negative when DNA was introduced from other Entamoeba species, including E. dispar, E. nuttalli and E. moshkovskii. Sequencing the PCR amplification fragment of the species specific primers produced a 134-nucleotide base sequence. The obtained DNA sequence was submitted for BLAST searching in Gen-Bank and the results showed that this determined sequence was the part sequence of E. histolytica peroxiredoxin gene. It indicated that this case has been diagnosed as E. histolytica naturally infecting in bat. No amoeba was detected in the fecal sample on days 5, 10 and 30 after the treatment.

DISCUSSION

Microscopic diagnosis is an important method for detecting the intestinal protozoan parasites. However, this method is incapable of distinguishing the pathogenic species E. histolytica from the nonpathogenic species E. dispar and the amphizoic amoeba E. moshkovskii by the morphology of cysts and trophozoites. The amplification of amoeba DNA fragments by Polymerase Chain Reaction (PCR) has proved to constitute a sensitive and specific method to detect and differentiate E. histolytica or E. dispar from the feces (Verweij et al., 2004; Solaymani-Mohammadi et al., 2006; Rafiei et al., 2009). Mere use of primers p11 plus p12 in PCR and sequencing is not enough to conclude that the isolate was indeed E. histolytica. Tachibana et al. (2007) proposed that an Entamoeba sp. strain from rhesus monkey is E. nuttalli. The primers p11 plus p12 used in this study can amplify the monkey isolates (Takano et al., 2007; Tachibana et al., 2007; Haghighi et al., 2008). So, a different marker (a 134-bp fragment inside the 16S-like small-subunit rRNA gene of E. histolytica (Gene Bank accession number X64142) be used to clearly identify the species of the Entamoeba isolate in this study. In this study, through observing the morphology, measuring amoeba trophozoites and cysts, using E. histolytica stool antigen detection kits, two PCR assays and DNA sequence analysis, the isolated strain was diagnosed as E. histolytica.

CONCLUSION

The results of present study suggest that amoebiasis of bat should be monitored more closely in the future.